Ecological characterization of Steinernema anatoliense (Rhabditida: Steinernematidae)

Our study describes the basic ecological characteristics of the entomopathogenic nematode Steinernema anatoliense including its response to temperature, moisture, and host range. The effect of temperature and soil moisture on the infection of Galleria mellonella larvae by S. anatoliense was determined. The temperature range for infectivity was greater than that for development. The optimal temperature for infection and development was 25 degrees C. Although S. anatoliense infected the hosts at 10 degrees C, no reproduction occurred at this temperature. This nematode species that was isolated from a cold region of Turkey exhibited warm-adapted temperature characteristics. Optimum water content of the soil for S. anatoliense to infect the host was 10%.     

Comparative study on the susceptibility of cutworms (Lepidoptera: Noctuidae) to Agrotis segetum nucleopolyhedrovirus and Agrotis ipsilon nucleopolyhedrovirus

The common cutworm (Agrotis segetum) and the black cutworm (Agrotis ipsilon) are serious soil pests of many vegetable and field crops all over the world. We have demonstrated the cross-infectivity of two baculoviruses, A. segetum nucleopolyhedrovirus (AgseNPV) and A. ipsilon nucleopolyhedrovirus (AgipNPV) for these two insect pests. The susceptibility of A. segetum to AgipNPV was confirmed by DNA restriction endonuclease analyses of DNA isolated from virus harvested from infected A. segetum larvae. For an initial comparison of both viruses, partial polyhedrin sequences were amplified by PCR, cloned, and sequenced. Both viruses shared a very similar polyhedrin gene sequence resulting in only three amino acid substitutions. Phylogenetic analyses clearly demonstrated that both viruses belong to NPV group II and are most closely related to a clade consisting of Spodoptera exigua NPV, Spodoptera frugiperda NPV, and Spodoptera littoralis NPV. Since AgipNPV shows high virulence for both cutworm species, it appears to be a suitable candidate as a single biological control agent of A. segetum and A. ipsilon.     

A Novel Strain of Steinernema riobrave (Rhabditida: Steinernematidae) Possesses Superior Virulence to Subterranean Termites (Isoptera: Rhinotermitidae)

Subterranean termites are major global pests of wood structures and wood products. Among the most economically important subterranean termite species in the US are Heterotermes aureus, Reticulitermes flavipes, and Coptotermes formosanus. In prior studies, the entomopathogenic nematode, Steinernema riobrave strain 355, exhibited a high level of virulence to H. aureus compared with other nematode species. However, S. riobrave 355 was reported to be poorly or only moderately virulent to R. flavipes and C. formosanus, respectively. We hypothesized that other strains of S. riobrave may possess a high level of virulence to all three termite species. Under laboratory conditions we compared three novel strains of S. riobrave (3-8b, 7-12, and TP) with the 355 strain for virulence to H. aureus, R. flavipes, and C. formosanus workers. H. aureus was very susceptible to all the S. riobrave strains, and termites in all nematode treatments were dead after 4 d. The TP strain of S. riobrave caused greater mortality in R. flavipes and C. formosanus compared to the other nematode strains. Specifically, the TP strain caused 75% and 91% mortality in R. flavipes and C. formosanus, respectively, which was more than 300% and 70% higher than the mortality caused by other strains. Additional studies are warranted to determine the ability of S. riobrave (TP) to control the targeted termite species under field conditions.     

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.     

Steinernema scapterisci n. sp. (Rhabditida: Steinernematidae)

Steinernema scapterisci n. sp., isolated in Uruguay from the mole cricket Scapteriscus vicinus, can be distinguished from other members in the genus by the presence of prominent cheilorhabdions, an elliptically shaped structure associated with the excretory duct, and a double-flapped epitygma in the first-generation female. The spicules of the male are pointed, tapering smoothly to a small terminus, and the shaft (calomus) is long, bearing a sheath. The gubernaculum has a long, upward-bent anterior part. The ratio of head to excretory pore divided by tail length of the third-stage juvenile is greater for S. scapterisci n. sp. than for S. carpocapsae. Steinernema scapterisci n. sp. did not hybridize with S. carpocapsae strain Breton. In laboratory tests, S. scapterisci n. sp. killed 10% or less of non-orthopteran insects, including the wax moth larva, a universal host for other species of Steinernema.     

Ecological aspects of an isolate of Steinernema diaprepesi (Rhabditida: Steinernematidae) from Argentina

Ecological aspects of Steinernema diaprepesi isolate SRC were studied to evaluate the species potential as biological control agent of insect pests. Under laboratory conditions, the following aspects were determined: the nematode life cycle, pathogenicity to several arthropods, reproductive capacity, tolerance to desiccation, effect of temperature on survival and infectivity of infective juveniles (IJs), and influence of soil texture and soil water potential on the isolate. The parasitic cycle on last-instar larvae of Galleria mellonella at 25°C was completed 8 days after infection. The nematode showed high virulence to lepidopteran larvae, being limited or nil in the remaining orders of arthropods evaluated. An acceptable offspring production of S. diaprepesi was confirmed in the species G. mellonella and S. frugiperda, suggesting that the isolate would have potential for control of lepidopteran larvae. Optimum temperature for reproduction was 20–25°C. IJs survived exposure to a range of temperatures between 10 and 40°C, with a significant reduction in the number of live IJs at 40°C. The nematodes remained infective at 20–40°C. IJ mortality was 100% on day 6 of exposure to 85% RH. The movement of IJs observed in the soil column experiments revealed that the isolate uses a cruiser-type search strategy. Soil texture and water potential significantly influenced IJ movement, search and penetration of G. mellonella larvae. The efficacy of this isolate was found to be favoured in sandy soils, regardless of the soil water potential.     

Molecular characterization of Agrotis segetum nucleopolyhedrovirus from Poland

The turnip moth, Agrotis segetum (Lepidoptera, Noctuidae), is an important pest insect in Europe, Asia, and Africa. We have genetically characterized and classified a nucleopolyhedrovirus isolated from A. segetum larvae in Poland (AgseNPV-P). The restriction pattern of AgseNPV-P was distinct from an isolate from England/France (AgseNPV-UK and AgseNPV-F). Sequence analysis of three conserved baculovirus genes, polyhedrin, lef-8 and pif-2, revealed that AgseNPV-P differs substantially from the already described NPVs isolated from A. segetum and possibly represents a new NPV species. Phylogenetic analysis placed AgseNPV-P among group II NPVs and showed the closest relationship to Agrotis ipsilon (Agip) NPV and Spodoptera exigua (Se) MNPV.     

Fine-structural characteristics of the antennal sensilla of Agrotis segetum (Insecta: Lepidoptera)

Summary The turnip moth Agrotis segetum possesses seven different types of sensilla: four single-walled (SW), one double-walled (DW), one terminal-pore (TP), and one poreless sensilla (NP).The SW 1 and SW 2 sensilla have the same external appearance, being long and slender, but differ in the branching pattern of the sensory processes: unbranched and branched in SW 1 and SW 2, respectively. The SW 3 sensilla are shorter, sickle-shaped, and contain a large number of branches from the sensory processes. These three sensillar types are innervated by 2–3 sensory cells. The SW 4 sensilla are raisin-shaped and possess three profusely branched sensory processes. The DW sensilla are short and have apical slit-like pores. This sensillar type has 5–6 sensory processes. The TP sensilla possess five sensory processes, one of them terminates basally in a tubular body, the others in the apical part of the long cuticular bristle. The NP sensilla are stout and have apical conelike structures. Two of the sensory processes terminate in the apical part, the third proximally. The third sensory process has a lamellar pattern. The fine structure indicates the following functions: SW and DW sensilla: chemoreception; TP sensillum: chemoreception and mechanoreception; NP sensillum: thermoreception and hygroreception.Supported by joint grants from the Swedish Council for Planning and Coordination of Research, the Swedish Natural Science Research Council, and the Swedish Council for Forestry and Agricultural Research     

Effect of Entomopathogenic Nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) on Meloidogyne mayaguensis Rammah and Hirschmann (Tylenchida: Meloidoginidae) Infection in Tomato Plants

Some studies suggest that entomopathogenic nematodes (EPN) affect plant-parasitic nematode populations. Here, the effects of live and dead IJ of Heterorhabditis bacteriophora JPM4, H. baujardi LPP7, Steinernema feltiae SN and S. carpocapsae All were evaluated against eggs and J2 of the plant-parasitic nematode Meloidogyne mayaguensis. According to treatment, 100 IJ were applied with 350 eggs, 350 J2 or 175 eggs + 175 J2 to tomato plants. Bioassays were conducted in March to May and repeated in September to November 2005. Both experiments lasted 9 weeks, and the variable evaluated was number of galls per plant. When eggs were used for infections in the first trial, plants exhibited lower gall number compared to control when live and dead H. baujardi IJ and live S. feltiae IJ were added (9.7, 4.5, 7.3 and 85.7 galls, respectively). In the second trial, live S. feltiae and S. carpocapasae IJ influenced gall formation compared to control (14.33, 14.57 and 168.02 galls, respectively). When J2 were used for infections, plants with live H. baujardi IJ presented less galls when compared to control in both trials (38.3 and 355.7 galls in the first trial and 145.2 and 326.2 in the second one, respectively). Infection with a mixture of J2 and eggs resulted in fewer galls than when live S. feltiae IJ were present in both trials, compared to control (38.3 and 44.2 galls vs. 275.3 and 192.2 galls, respectively). We conclude that H. baujardi and S. feltiae apparently may be inhibiting egg hatching and J2 infection.     

Mating Behavior and Reproductive Potential in the Turnip Moth Agrotis segetum (Lepidoptera: Noctuidae)

We investigated the lifetime mating potential and the reproductive behavior of male and female turnip moths Agrotis segetum (Schiff.) under field and laboratory conditions. The sex ratio was 1 : 1 in a lab-reared population as well as in two wild populations. Males were capable of mating repetitively a relatively large number of times (mean of 6.7 ± 2.7 matings) when given access to new virgin females throughout their lifetimes. Females seldom mated more than once (mean ± 1.3 ± 0.6 matings), indicating a male-biased operational sex ratio. The mean potential lifetime mating was five times higher in males, while the coefficient of variance was lower in males. There was no differences in longevity between animals that were allowed to mate and animals not allowed to mate, indicating no direct costs or benefits of mating in physiological terms. In males, the number of matings was positively correlated with longevity, but this was not the case in females. Nor was there a correlation between the number of female matings and the number of fertilized eggs. There was a negative correlation between the number of eggs fertilized and the number of times males had previously mated, indicating that male ejaculates were limited. Male spermatophore size also decreased with number of achieved matings. Laboratory-reared females attracted males in the field throughout their lifetimes, with a peak at 3–7 days of age. Wild males, allowed to choose between pairs of caged females in the field, were attracted in equal numbers to females of different ages. Females did not show any mate-rejection behavior in the field. They mated with the first male that courted them. No incidence of mate replacement by males arriving later to already courted females were recorded.     

Identification of Entomopathogenic Nematodes in the Steinernematidae and Heterorhabditidae (Nemata: Rhabditida)

This paper contains taxonomic keys for the identification of species of the genera Steinernema and Heterorhabditis. Morphometrics of certain life stages are presented in data tables so that the morphometrics of species identified using the keys can be checked in the tables. Additionally, SEM photographs and diagnoses of the families and genera of Steinernematidae and Heterorhabditidae are presented.     

Assessing Nuclear and Mitochondrial DNA Sequence Variation Within Steinernema (Rhabditida: Steinernematidae)

DNA sequence analysis was used to characterize the nuclear ribosomal DNA ITS1 region and a portion of the COII and 16S rDNA genes of the mitochondrial genome from Steinernema entomopathogenic nematodes. Nuclear ITS1 nucleotide divergence among seven Steinernema spp. ranged from 6 to 22%, and mtDNA divergence among five species ranged from 12 to 20%. No intraspecific variation was observed among three S. feltiae strains. Phylogenetic analysis of both nuclear and mitochondrial DNA sequences confirms the existing morphological relationships of several Steinernema species. Both the rDNA ITS1 and mtDNA sequences were useful for resolving relationships among Steinernema taxa.     

Steinernema akhursti sp. n. (Nematoda: Steinernematidae) from Yunnan, China

A new species of entomopathogenic nematode, herein described as Steinernema akhursti sp. n., was recovered from soil samples collected from Yunnan Province, the People's Republic of China. Both morphological and molecular data show congruently that S. akhursti sp. n. belongs to the Steinernema feltiae group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For the first generation male, the new species can be recognized by spicule length 90 +/- 4.6 microm, spicule tip blunt with an aperture on the ventral side, gubernaculum with a long and needle-shaped cuneus, and tail conoid with a prominent mucron on the tip and a concave on ventral side. For the infective juvenile, the combination of the following characters: body length 812 +/- 19 microm, distance from anterior end to excretory pore 59 +/- 1.5 microm, tail length 73 +/- 2.9 microm, E% 77 +/- 4.5, lateral field with six evenly distributed and identical ridges at the middle body portion, and tail with long and slightly constrict hyaline portion can be used to separate the new species from other nematodes. For the female, the new species is characterized by: tail conoid with a short mucron and slightly swelling anal portion and a symmetrical, slightly protruding vulva with conspicuous double-flapped epiptygma. The nematode can be separated from other described species of Steinernema by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA and from the closely related species S. feltiae and Steinernema oregonense by cross-breeding tests.     

Steinernema neocurtillis n. sp. (Rhabditida: Steinernematiclae) and a Key to Species of the Genus Steinernema

Steinernema neocurtillis n. sp. isolated from the mole cricket Neocurtilla hexadactyla Perty can be distinguished from other members of the genus by characteristics of the first-generation male and the third-stage infective juvenile (IJ). In the male, the distance from the anterior end to the excretory pore (DAE) is less than the body width at the excretory pore; D% (DAE divided by length of esophagus x 100) is low at 19. The gubernaculum legth is greater than three-fourths the spicule length. Range of the ratio gubernaculum length divided by spicule length is 0.82-0.93 in the first-generation male and 0.92-1.00 in the second-generation male. In the IJ, the distance from the anterior end to the excretory pore is extremely short (18 μm), causing the D% and E% (DAE divided by tail length x 100) to be low (D% = 23 and E% = 12). Average body length of the IJ is 885 μm.     

Morphological and Ecological Characterization of Steinernema feltiae (Rhabditida: Steinernematidae) Rioja Strain Isolated from Bibio hortulanus (Diptera: Bibionidae) in Spain

A new strain of Steinernema feltiae (Rhabditida: Steinernematidae) was isolated in La Rioja (Spain) from larvae of Bibio hortulanus (Diptera: Bibionidae). A comparative morphometric analysis of this new strain and four additional S. feltiae isolates was performed. Although significant differences in morphometric measurements were observed, PCR-RFLP profiles and sequence analysis of the ITS region of rDNA confirmed the identity of the new strain as A2 RFLP type of S. feltiae. A comparative morphometric study among nematodes from three hosts, Galleria mellonella (Lepidoptera: Pyralidae), Spodoptera littoralis (Lepidoptera: Noctuidae) and B. hortulanus, was conducted. Ecological characterization of the Rioja isolate was performed in G. mellonella larvae. Larval mortality was 75.3 and 78.12% in penetration and sand column assays, respectively, and the percentage of penetrating infective juveniles was 12.0 and 2.8% in these assays. Larval mortality in the one-on-one bioassay was 4.2%, and in exposure-time bioassays, it was 50% at 11.25 hours. Relationships between morphometric characteristics and host mortality are discussed for this new strain of entomopathogenic nematode.     

Fruit Bins Washed with Steinernema carpocapsae (Rhabditida: Steinernematidae) to Control Cydia pomonella (Lepidoptera: Tortricidae)

A bin washing apparatus (drencher) was used to treat late-instar codling moth, Cydia pomonella Linnaeus-infested wooden fruit bins with the entomopathogenic nematode Steinernema carpocapsae . Sentinel late-instar codling moth larvae were placed in strips of wood, made from dismantled bins, that were subsequently screwed into the corners of real bins before treatment. Pre-soaking the bins with water prior to treatment and covering the bins with a plastic tarp post-treatment, to maintain a high humidity, significantly increased sentinel codling moth mortality. Treatment for 1 min with 50 S. carpocapsae infective juveniles mL -1 water resulted in more than 80% mortality over 6 h in which the nematodes were continuously pumped through the drencher. The temperature of the water in the bin washer was ≤15°C and the chlorine in the city water was too low to harm the nematodes. The key disadvantage of washing the bins was the labour involved. It may be possible to reduce post-treatment holding time and increase codling moth mortality by holding the bins at a higher temperature post-treatment, however this parameter was not included in the study. The presence of the codling moth granulosis virus within the larvae did not influence the nematode-induced codling moth mortality over a range of concentrations and two temperatures in laboratory trials.     

The significance of shelter for young cutworms (Agrotis segetum)

The importance of shelter for young Agrotis segetum Schiff. (Lep. Noctuidae) larvae was investigated using caged carrots (Daucus carota L.) with the soil surface covered only by paraffin wax, or with a layer of vermiculite resembling dry soil on top of a layer of paraffin wax. In the vermiculite treatment, most cutworms were found in the vermiculite with only a small proportion on the plant foliage, whereas in the paraffin wax treatment, the majority was found on the foliage. Larval growth rate was higher and mortality lower among the cutworms which could shelter in the vermiculite than amongst those with no shelter. These results agree with those from previous investigations which showed that moist soil prevented the larvae from sheltering in it. The importance of shelter in dry soil cannot be fully explained, but it may provide a more favourable temperature for larval development.

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Résumé L'importance des abris pour les jeunes chenilles d'A. segetum a été examinée en utilisant des carottes (Daucus carota) dans des cages dont le sol était recouvert d'une couche de paraffine ou d'une couche de vermiculite mimant du sol sec au-dessus de la couche de paraffine. La plupart des vers gris ont été trouvés dans la couche de vermiculite contre une faible proportion dans le feuillage, tandis qu'en absence de vermiculite la majorité a été retrouvée dans le feuillage. La croissance larvaire était meilleure et la mortalité plus faible avec la vermiculite. Ces résultats sont conformes à des observations antérieures qui avaient montré qu'un sol humide empechait les chenilles de s'y abriter. L'importance de l'abri en sol sec n'est pas entièrement expliquée, mais celui-ci pourrait assurer une température plus favorable au développement larvaire.

     

Steinernema diaprepesi n. sp. (Rhabditida: Steinernematidae), a Parasite of the Citrus Root Weevil Diaprepes abbreviatus (L) (Coleoptera: Curculionidae)

A nematode collected from Diaprepes abbreviatus is identified and described as a new species, Steinernema diaprepesi n. sp. The new species is closely related to S. feltiae, S. glaseri, and S. oregonense and can be distinguished from these species by the following characteristics: Males: Spicule averaging 79 (71-90) µm and spicule shape; D% (distance from anterior end to excretory pore/ esophagus length × 100) about 80; the ratio SW (spicule length/anal body width) about 1.8. Females: Vulva with short, double- flapped epiptygma; tail terminus usually with 5 papillae-like structures. Infective juveniles: Body averaging 1,002 (880-1,133) µm, EP (distance from anterior end to excretory pore) = 74 (66-83) µm; tail length = 83 (65-91) µm, and E% (EP/tail length × 100) = 89.6 (78-114). Lateral field pattern variable, the formula for the arrangement of ridges from head to tail is: 2, 6, 7, 8, 4, 2. The portion with eight ridges is the longest. This new species can be differentiated further from three closest species (S. feltiae, S. glaseri, and S. oregonense) by characteristic sequences of their ITS regions, including sequence lengths, ratios of similarity, composition, and differences in base characters in sequence alignment.     

Interactions of two idiobiont parasitoids (Hymenoptera: Ichneumonidae) of codling moth (Lepidoptera: Tortricidae) with the entomopathogenic nematode Steinernema carpocapsae (Rhabditida: Steinernematidae)

Simultaneous use of parasitoids and entomopathogenic nematodes for codling moth (CM) control could produce an antagonistic interaction between the two groups resulting in death of the parasitoid larvae. Two ectoparasitic ichneumonid species, Mastrus ridibundus and Liotryphon caudatus, imported for classical biological control of cocooned CM larvae were studied regarding their interactions with Steinernema carpocapsae. Exposure of M. ridibundus and L. caudatus developing larvae to infective juveniles (IJs) of S. carpocapsae (10 IJs/cm2; approximately LC(80-90) for CM larvae) within CM cocoons resulted in 70.7 and 85.2% mortality, respectively. However, diapausing full grown parasitoid larvae were almost completely protected from nematode penetration within their own tightly woven cocoons. M. ridibundus and L. caudatus females were able to detect and avoid ovipositing on nematode-infected cocooned CM moth larvae as early as 12h after treatment of the host with IJs. When given the choice between cardboard substrates containing untreated cocooned CM larvae and those treated with an approximate LC95 of S. carpocapsae IJs (25 IJs/cm2) 12, 24, or 48h earlier, ovipositing parasitoids demonstrated a significant preference for untreated larvae. The ability of these parasitoids to avoid nematode-treated larvae and to seek out and kill cocooned CM larvae that survive nematode treatments enhances the complementarity of entomopathogenic nematodes and M. ridibundus and L. caudatus.     

Effect of Soil Depth and Moisture on the Vertical Distribution of Steinernema riobrave (Nematoda: Steinernematidae)

The effect of soil moisture on the distribution of Steinernema riobrave in a sand column was determined. Larvae of Pectinophora gossypiella were used to detect S. riobrave infective juveniles (IJ) in each 2.5-cm section of 30-cm-long soil columns. Soil moisture was determined for each section and related to the numbers of nematodes recovered from infected insect baits. Infective juveniles of S. riobrave applied on the sand column surface showed some degree of positive geotaxis. IJ in soil columns with a consistent moisture gradient grouped in the upper 12.7 cm within a water potential range of ¯40 to ¯0.0055 MPa (2% to 14% moisture). Nematodes in sand columns that were gradually dehydrating moved down the soil column, aggregating on the 28th day between 15-23 cm in depth. Nematode redistribution over time allowed IJ to remain within a water potential range of ¯0.1 to ¯0.012 MPa (5.2% to 9.5% moisture).