Evaluation of different sponge types on the survival and infectivity of stored entomopathogenic nematodes.

Sponges are one of the cheapest and most suitable substrates used to formulate and/or store the infective juveniles (IJs) of entomopathogenic nematodes (EPNs). Our study investigated the survival and infectivity of the IJs on five different sponges compared to that in an aqueous suspension (control). The sponges were Oasis® floral, Nanosponge, ScotchbriteTM, or Lysol® and natural sea sponge. EPN species tested were Heterorhabditis bacteriophora, Steinernema carpocapsae and S. feltiae. The recovery efficiency of the IJs from sponges was initially assessed. Subsequently, IJs were stored in the sponges and placed in plastic bags or Falcon tubes and incubated at 10° or 27 °C for 8 months or 11 weeks, respectively. IJ survival and infectivity were monitored monthly for the 10 °C and weekly for 27 °C in these sponge types. The IJs were recovered from the sponges, and their survival was based on observing their movement under a dissecting microscope, and infectivity was based on larval mortality in Galleria mellonella. Recovery efficiency of IJs was best for the Oasis floral sponge for all nematode species ranging between 83 and 91%. The survival and infectivity of stored IJs in all sponge types and control for both 10° and 27 °C gradually decreased over time. IJs stored in Scotchbrite, Lysol, and Nanosponge had the best survival and infectivity, whereas Oasis floral and natural sea sponges showed the poorest results. After 8 months at 10 °C in plastic bags, the survival ratio of all IJs in these three sponges (Scotchbrite, Lysol, and Nanosponge) was approximately 55%. IJs in Scotchbrite and Nanosponge were also able to survive and retain their infectivity at 27 °C for 3 months. IJs stored in Falcon tubes had survival that ranged from 26 to 53% at 27 °C and 55 to 77% at 10 °C. H. bacteriophora IJs lost their infectivity when stored at 27 °C after 10 weeks. However, S. carpocapsae and S. feltiae exhibited 85% infectivity when stored in Scotchbrite and 50% in Nanosponge, respectively. Overall, sponges made from polyurethane (Scotchbrite) followed by melamine (Nanosponge) and cellulose (Lysol) are recommended for long-term nematode storage and transportation of nematode samples. However, Oasis floral sponge may be preferred for short-term IJ formulation for field applications because of easier recovery of IJs.

Response of three cyprinid fish species to the Scavenger Deterrent Factor produced by the mutualistic bacteria associated with entomopathogenic nematodes.

The symbiotic bacteria, Photorhabdus and Xenorhabdus associated with entomopathogenic nematodes (EPNs) in the genera Heterorhabditis and Steinernema, respectively, produce a compound(s) called the Scavenging Deterrent Factor (SDF). SDF deters a number of terrestrial insect scavengers and predators and one bird species from feeding on host insects killed by the nematode-bacterium complex but has not been tested against aquatic vertebrates. Moreover, the Heterorhabditis-Photorhabdus association is believed to have evolved in an aquatic environment. Accordingly, we hypothesized that SDF will deter fish from feeding on nematode-killed insects and tested the responses of three omnivorous fresh water fish species, Devario aequipinnatus, Alburnoides bipunctatus, and Squalius pursakensis, to SDF in the laboratory. When the fish were exposed to Galleria mellonella larvae killed by the Heterorhabditis- or Steinernema-bacterium complex at 2 or 4days post-infection, all three fish species made several attempts to consume the cadavers but subsequently rejected them. However, all fish species consumed freeze-killed control larvae. In a choice test, when D. aequipinnatus or A. bipunctatus were offered a pair of nematode-killed larvae, both fish species rejected these cadavers; when offered a nematode-killed larva and a freeze-killed larva, both fish species consumed the freeze-killed larva but not the nematode-killed one. In further tests with D. aequipinnatus, there was no significant difference in the number of 2-day-old Bacillus thuringiensis subsp. kurstaki-killed (Btk) larvae consumed compared to freeze-killed larvae, but significantly fewer 4-day-old Btk-killed larvae were consumed compared to freeze-killed larvae. When D. aequipinnatus was fed G. mellonella larvae killed by the symbiotic bacteria, the fish rejected the cadavers. When given freeze-killed or nematode-killed mosquito (Aedes aegypti) larvae, the fish consumed significantly more of the former larvae (99%) compared to the latter (55%). When D. aequipinnatus was placed in a symbiotic cell-free supernatant for 18h, a significant reduction in consumption of freeze-killed larvae compared to cell-free Btk or control broth supernatant was observed. We showed that SDF protects the nematode-killed insects from being consumed by omnivorous fishes and suggests that they will have minimal effects on recycling of EPNs in the aquatic environment.

A laboratory study on the effect of Paraiotonchium autumnale parasitism on the longevity of Musca autumnalis.

Paraiotonchium autumnale (Nickle) (Tylenchida: Iotonchiidae) causes parasitic sterilization in female Musca autumnalis DeGeer (Diptera: Muscidae). In addition to sterilization, P. autumnale causes physiological and behavioural changes within its host. However, there have been no records of reduced host lifespan in this system. Studies were conducted in 2009 and 2010 with wild M. autumnalis collected as larvae from cow dung pats from Browns Valley, California, USA. Field-collected larvae were reared to adulthood and flies of the same eclosion dates were caged together, regardless of whether or not they were parasitized. Dead flies were collected daily, and parasitism status was confirmed by dissection. Due to the very different effects of P. autumnale-parasitism on male versus female face flies, flies were analysed separately by sex. Kaplan-Meier analysis revealed a non-significant difference in longevity between non-parasitized and parasitized flies for female and male flies in 2009. In 2010, however, significant differences (P < 0·05) were found between parasitized and non-parasitized female and male flies. In 2010, the median time to death for non-parasitized female flies was 20 days and 15 days for parasitized females. The 2010 median survival time for non-parasitized male flies was 33 days and 15 days for parasitized males. This is a parasite-mediated cost to the host that has not been recorded previously. Based on our results and results from another published study we suggest that the potential for sterilizing parasites to alter host longevity is condition dependent.

Scavenger deterrent factor (SDF) from symbiotic bacteria of entomopathogenic nematodes.

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are symbiotically associated with bacteria in the genera Xenorhabdus and Photorhabdus, respectively. The symbiotic bacteria produce a chemical compound(s) that deterred ants from feeding on nematode-killed insects (i.e., cadavers) and has been previously referred to as an Ant Deterrent Factor (ADF). We studied the response of different arthropod scavenger species which included the ant Lepisiota frauenfeldi, cricket Gryllus bimaculatus, wasps Vespa orientalis and Paravespula sp., and calliphorid fly Chrysomya albiceps, to ADF. These scavengers (ants, crickets, and wasps) were exposed to cadavers with and without the nematode/bacterium complex or to Photorhabdus luminescens cultures of different ages on different substrates. The ant, cricket, and wasp species did not feed on nematode-killed insects containing the nematode/bacterium complex that were 2 days old and older but fed on 1-day-old nematode-killed and freeze -killed insects. Crickets consumed 2- to 7-day-old axenic nematode-killed insects, 1-, 4-, and 5-day-old insects killed by the bacterium, Serratia marcescens, and freeze-killed, putrid insects that were up to 10 days old. The crickets only partially consumed 2- and 3-day-old insects killed by S. marcescens which differed significantly from the 1-, 4-, and 5-day-old killed insects by this bacterium. Ants fed only on 5% sucrose solution (control) and 1- to 3- day old cultures of P. luminescens containing 5% sucrose but not on older cultures of P. luminescens. Wasps did not feed on meat treated with P. luminescens supernatant, whereas they fed on meat treated with Escherichia coli supernatant and control meat. Calliphorid flies did not oviposit on meat treated with P. luminescens supernatant but did oviposit on untreated meat. Based on the response of these scavengers, the chemical compound(s) responsible for this deterrent activity should be called "scavenger deterrent factor" (SDF).

Life history of Sancassania polyphyllae (Acari: Acaridae) feeding on dissected tissues of its phoretic host, Polyphylla fullo (Coleoptera: Scarabaeidae): temperature effects.

The effect of temperature on the development and fecundity of Sancassania polyphyllae fed on tissues of Polyphylla fullo larvae was studied at 15, 20, 25, 30, and 35 ± 1°C and 65 ± 10% RH in a dark incubator. Mean developmental period of immature stages decreased significantly with increasing temperatures from 15 to 30°C. Developmental periods at 30-35°C were not significantly different. The estimated lower developmental thresholds of the various immature stages ranged between 10.1 and 11.5°C. The thermal constant for the egg-to-female adult was 93.5 degree-days. The pre-oviposition, oviposition, and post-oviposition periods and female longevity were significantly longer at 15°C than at higher temperatures. Mean total and daily fecundity were the highest at 25°C, which were significantly different from those obtained at 15, 20 and 30°C. The net reproductive rate (R (0)) was the highest at 25°C (588.3 ♀/♀). The longest mean generation time (T (0)) occurred at 15°C (36 days) and the shortest occurred at 30°C (9.2 days). The highest intrinsic rate of increase (r (m)) for S. polyphyllae was observed at 25 (0.61 ♀/♀/day) and 30°C (0.62 ♀/♀/day).

Temperature effects on Korean entomopathogenic nematodes, Steinernema glaseri and S. longicaudum, and their symbiotic bacteria.

We investigated the temperature effects on the virulence, development, reproduction, and motility of two Korean isolates of entomopathogenic nematodes, Steinernema glaseri Dongrae strain and S. longicaudum Nonsan strain. In addition, we studied the growth and virulence of their respective symbiotic bacterium, Xenorhabdus poinarii for S. glaseri and Xenorhabdus sp. for S. longicaudum, in an insect host at different temperatures. Insects infected with the nematode-bacterium complex or the symbiotic bacterium was placed at 13 degrees C, 18 degrees C, 24 degrees C, 30 degrees C, or 35 degrees C in the dark and the various parameters were monitored. Both nematode species caused mortality at all temperatures tested, with higher mortalities occurring at temperatures between 24 degrees C and 30 degrees C. However, S. longicaudum was better adapted to cold temperatures and caused higher mortality at 18 degrees C than S. glaseri. Both nematode species developed to adult at all temperatures, but no progeny production occurred at 13 degrees C or 35 degrees C. For S. glaseri, nematode progeny production was best at inocula levels above 20 infective juveniles/host at 24 degrees C and 30 degrees C, but for S. longicaudum, progeny production was generally better at 24 degrees C. Steinernema glaseri showed the greatest motility at 30 degrees C, whereas S. longicaudum showed good motility at 24 degrees C and 30 degrees C. Both bacterial species grew at all tested temperatures, but Xenorhabdus sp. was more virulent at low temperatures (13 degrees C and 18 degrees C) than X poinarii.

Predation of entomopathogenic nematodes by Sancassania sp. (Acari: Acaridae).

Predation of the entomopathogenic nematode, Steinernema feltiae (Rhabditida: Steinernematidae), by Sancassania sp. (Acari: Acaridae) isolated from field-collected scarab larvae was examined under laboratory conditions. Adult female mites consumed more than 80% of the infective juvenile (IJ) stage of S. feltiae within 24 h. When S. feltiae IJs were exposed to the mites for 24 h and then exposed to Galleria mellonella (Lepidoptera: Pyralidae) larvae, the number of nematodes penetrating into the larvae was significantly lower compared to S. feltiae IJs that were not exposed to mites (control). Soil type significantly affected the predation rate of IJs by the mites. Mites preyed more on nematodes in sandy soil than in loamy soil. We also observed that the mites consumed more S. feltiae IJs than Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae). No phoretic relationship was observed between mites and nematodes and the nematodes did not infect the mites.

Distribution and adult activity of Popillia quadriguttata (Coleoptera: Scarabaeidae) on golf courses in Korea.

Japanese beetle traps baited with the Popillia japonica Newman (Coleoptera: Scarabaeidae) pheromone lure and a eugenol feeding attractant were placed at five golf courses in Korea to determine how well they work for detecting activity of a closely related species, Popillia quadriguttata (F.) (Coleoptera: Scarabaeidae), a turf pest in Korea. The traps also were used to determine the time of day and time of year that P. quadriguttata is most active. Nineteen scarab species of 13 genera were attracted to the Japanese beetle traps with P. quadriguttata clearly being the most abundant (383 beetles per trap), followed by Adoretus tenuimaculatus Waterhouse (10 per trap), Popilliaflavosellata Fairmaire (seven per trap), Exomala orientalis Waterhouse (four per trap), and Maladera japonica (two per trap). Other scarab species were trapped at a rate of <1.0 per trap. Popillia quadriguttata adults were active over a 5-wk period in late June and early July. At Yongwon Golf Club in 2002, peak adult activity was during the last week of June in visual counts and approximately 1 wk later in the Japanese beetle traps. In Korea, P. quadriguttata adults are most active between 12:00 p.m. and 4:00 p.m. This information should be helpful to golf course superintendents in Korea and to entomologists interested in finding natural enemies of P. quadriguttata to evaluate as potential biocontrol organisms for the very closely related species, the Japanese beetle.

Dispersal, Infectivity and Sex Ratio of Early- or Late-Emerging Infective Juveniles of the Entomopathogenic Nematode Steinernema carpocapsae.

Differences in activity between infective juveniles (IJ) of the entomopathogenic nematode Steinernema carpocapsae that emerged directly from cadavers onto either a sand or agar substrate compared with those emerging from a cadaver into water and then being placed on the same substrate are known to occur. Differences between S. carpocapsae IJ that emerged directly from a cadaver vs. those that emerged from a cadaver and held in water were further elucidated. Dispersed and non-dispersed IJ from a cadaver were compared with those held in water between two time periods designated as early- (first two days) or late-emerging IJ (seventh day). A significantly greater proportion of early-emerging IJ from the cadaver treatment dispersed, compared with late-emerging IJ from a cadaver or either group of emerging IJ held in aqueous suspension. Moreover, IJ from cadavers were more infectious than those from the aqueous suspensions, and IJ that dispersed were less infectious than those that did not disperse. IJ that emerged early were mostly males, whereas those that emerged late were mostly females. For the non-dispersed IJ, most that emerged early were males, and those that emerged later were females, but among dispersing IJ, there was no difference in sex ratio between early- and late-emerging nematodes.

Definitions of pathogenicity and virulence in invertebrate pathology.

Accurate definition and usage of terminology are critical to effective communication in science. In a recently published article, the clarity and consistency of the terms pathogenicity and virulence as used in invertebrate pathology were called into question, and a revision of these terms was proposed. Our objective was to examine definitions of pathogenicity and virulence and their use in invertebrate pathology, and respond to this article. Although usage of the terms pathogenicity and virulence varies, we found considerable consistency in the published definitions of these terms in the invertebrate pathology literature throughout the history of the discipline, as well as among related disciplines such as medicine and microbiology. We did not find the established definitions to be lacking in clarity or utility. Therefore, we recommend that the definition and use of these terms adhere to precedence. Specifically, pathogenicity is the quality or state of being pathogenic, the potential ability to produce disease, whereas virulence is the disease producing power of an organism, the degree of pathogenicity within a group or species. Pathogenicity is a qualitative term, an "all-or-none" concept, whereas virulence is a term that quantifies pathogenicity.

Response of ants to a deterrent factor(s) produced by the symbiotic bacteria of entomopathogenic nematodes.

The production of an ant-deterrent factor(s) (ADF) by Xenorhabdus nematophila and Photorhabdus luminescens, the symbiotic bacteria of the nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora, respectively, was examined. In addition to an in vivo assay in which bacteria were tested for their ability to produce ADF within insect cadavers (M.E. Baur, H. K. Kaya, and D. R. Strong, Biol. Control 12:231-236, 1998), an in vitro microtiter dish assay was developed to monitor ADF activity produced by bacteria grown in cultures. Using these methods, we show that ADF activity is present in the supernatants of bacterial cultures, is filterable, heat stable, and acid sensitive, and passes through a 10-kDa-pore-size membrane. Thus, ADF appears to be comprised of a small, extracellular, and possibly nonproteinaceous compound(s). The amount of ADF repellency detected depends on the ant species being tested, the sucrose concentration (in vitro assays), and the strain, form, and age of the ADF-producing bacteria. These findings demonstrate that the symbiotic bacteria of some species of entomopathogenic nematodes produce a compound(s) that deters scavengers such as ants and thus could protect nematodes from being eaten during reproduction within insect cadavers.

Laboratory and field evaluation of Korean entomopathogenic nematode isolates against the oriental beetle Exomala orientalis (Coleoptera: Scarabaeidae).

The oriental beetle Exomala (Anomala) orientalis (Waterhouse) is an important pest of turfgrass in Korean golf courses, and although a few chemical insecticides are registered for insect pest control, they are not very effective against scarab larvae. There is also a growing concern in Korea about the run-off of insecticides into sensitive habitats and the potential for groundwater contamination. A safe and environmentally sound alternative is needed to conventional insecticides. We therefore evaluated six Korean entomopathogenic nematode isolates: S. carpocapsae Pocheon, S. glaseri Dongrae, S. glaseri Mungyeong, S. longicaudum Gongju, S. longicaudum Nonsan, and Heterorhabditis sp. Gyeongsan for their potential as bioinsecticides for control of E. orientalis. In addition, we evaluated a reduced chemical insecticide approach that combined chlorpyrifos-methyl with nematodes. In laboratory tests Heterorhabditis sp. Gyeongsan was the most efficacious, causing 100% mortality of the second and 38% of the third instars. All other nematode isolates caused 50-80% mortality of the second and 15-30% of the third instars. E. orientalis pupae were highly susceptible to all the Korean entomopathogenic nematode isolates except S. carpocapsae. In artificially infested field plots, all Korean nematode isolates cause 50-70% mortality of the third instar. A combination of a one-half rate of Heterorhabditis sp. and a one-half rate chlorpyrifos-methyl was synergistic, causing 91% mortality compared with 69% for the full rate of Heterorhabditis sp. or 22% for the full rate of chlorpyrifos-methyl. In a second field trial, a natural infestation of preoverwintering third instar was treated. In this trial a one-half rate of S. longicaudum Nonsan plus a one-half rate of chlorpyrifos-methyl caused 96.8% mortality, much more than a full rate of S. longicaudum Nonsan (45.9% mortality) or a full rate of chlorpyrifos-methyl (28.7% mortality). The interactions of Heterorhabditis sp. and S. longicaudum Nonsan with chlorpyrifos-methyl in our field trials appear to be synergistic.

Life history and spatial distribution of Oriental beetle (Coleoptera: Scarabaeidae) in golf courses in Korea.

Larval and adult activity of the oriental beetle Exomala orientalis (Waterhouse), a pest of turfgrass in Korea, was investigated at four golf clubs in Pusan, Korea, from 1995 to 1999. Adult emergence was first observed on the greens in late May with peak activity occurring 2 wk later. During the day, E. orientalis adults were most active between 1800 and 2200 hours. First instars were found mostly in early July, second instars mostly in late July, and third instars from August to April. The density of larvae in fixed plots decreased steadily from the time of egg laying to pupation: 667/m3 on 26 July, 267/m3 on 29 August, and 122/m3 on 2 October 1997. All the observed E. orientalis completed one generation per year. Adult females were observed feeding on flowers of a late-blooming variety of Japanese chestnut (Castanea crenata Sieb & Zucc). E. orientalis larval densities were higher in greens with Japanese chestnut nearby, and where magpie, Pica pica sericea (Gould), feeding was observed. More E. orientalis adults emerged from the right, left, and back of greens than from the front or middle. The intensity of emergence was inversely proportional to the amount of golfer traffic on various parts of the green. Counting emergence holes may be a way that golf course superintendents can predict which greens and tees are most likely to be damaged from E. orientalis larvae without destructive sampling.

Effects of Etomopathiogenic Nematodes on Meloidogyne javanica on Tomatoes and Soybeans.

Two Hawaiian isolates of Steinernema feltiae MG-14 and Heterohabditis indica MG-13, a French isolate of S. feltiae SN, and a Texan isolate of S. riobrave TX were tested for their efficacy against the root-knot nematode, Meloidogyne javanica, in the laboratory and greenhouse. Experiments were conducted to investigate the effects of treatment application time and dose on M. javanica penetration in soybean, and egg production and plant development in tomato. Two experiments conducted to assess the effects of entomopathogenic nematode application time on M. javanica penetration demonstrated that a single application of 10 S. feltiae MG-14 or SN infective juveniles per 100 cm(3) of sterile soil, together with 500 (MG-14) or 1,500 (SN) second-stage juveniles of M. javanica, reduced root penetration 3 days after M. javanica inoculation compared to that of a water treatment. Entomopathogenic nematode infective juveniles applied to assess the effects on M. javanica egg production did not demonstrate a significant reduction compared to that of the water control treatment. There was no dose response effect by Steinernema spp. On M. javanica root penetration or egg production. Steinernema spp. did not affect the growth or development of M. javanica-infected plants, but H. indica MG-13-treated plants had lower biomass than untreated plants infected with M. javanica. Infective juveniles of S. riobrave TX, S. feltiae SN, and MG-14 but not those of H. indica MG-13 were found inside root cortical tissues of M. javanica-infected plants. Entomopathogenic nematode antagonism to M. javanica on soybean or tomato was insufficient in the present study to provide a consistent level of nematode suppression at the concentrations of infective juveniles applied.

Virulence and site of infection of the fungus, Hirsutella thompsonii, to the honey bee ectoparasitic mite, Varroa destructor.

The Varroa mite, Varroa destructor, is recognized as the most serious pest of both managed and feral Western honey bee (Apis mellifera) in the world. The mite has developed resistance to fluvalinate, an acaricide used to control it in beehives, and fluvalinate residues have been found in the beeswax, necessitating an urgent need to find alternative control measures to suppress this pest. Accordingly, we investigated the possibility of using the fungus, Hirsutella thompsonii, as a biocontrol agent of the Varroa mite. Among the 9 isolates of H. thompsonii obtained from the University of Florida and the USDA, only the 3 USDA isolates (ARSEF 257, 1947 and 3323) were infectious to the Varroa mite in laboratory tests. The mite became infected when it was allowed to walk on a sporulating H. thompsonii culture for 5 min. Scanning electron micrographs revealed that the membranous arolium of the mite leg sucker is the focus of infection where the fungal conidia adhered and germinated. The infected mites died from mycosis, with the lethal times to kill 50% (LT(50)s) dependent on the fungal isolates. Thus, the LT(50)s were 52.7, 77.2, and 96.7h for isolates 3323, 257, and 1947, respectively. Passage of H. thompsonii through Varroa mite three times significantly reduced the LT(50)s of isolates 257 and 1947 (P<0.05) but not the LT(50) of isolate 3323. The fungus did not infect the honey bee in larval, prepupal, pupal, and adult stages under our laboratory rearing conditions. Our encouraging results suggest that some isolates of H. thompsonii have the potential to be developed as a biocontrol agent for V. destructor. However, fungal infectivity against the mites under beehive conditions needs to be studied before any conclusion can be made.