Survival and Persistence

The infective juveniles of parasitic nematodes are capable of surviving for weeks or even months without any exogenous source of energy (Croll and Matthews 1977). They depend exclusively on their accumulated energy reserves. Where conditions are otherwise favourable, the duration of survival is determined by the amount and rate of utilization of the reserves. Lipids especially neutral lipids are the main energy reserve (Barrett and Wright, 1998) and may account for a considerable proportion of the body contents. In the IJs of EPN Steinernema and Heterorhabditis, lipids constitute 32% and 43% of total body weight (Fitters et al., 1999; Selvan et al., 1993). There is evidence that starvation is the main cause of mortality for EPN IJ in water (Qiu and Bedding, 2000). Motility and infectivity have been correlated with depletion of reserves in EPN stored in water or moist sponge (Lewis et al., 1995; Patel et al., 1997; Vanninen 1990).

Depletion of energy reserves in soil has not been studied for any species though it is assumed that abiotic factors such as temperature, soil moisture and texture influence survival and infectivity partly by affecting the rate of utilization of reserves, especially the lipids (Kung et al., 1990;1991; Molyneux 1985). Hass et al., 2001 found that persistence of 10 strains of Heterorhabditis in soil was significantly correlated with their persistence in water, suggesting that the rate of utilization of energy reserves may be an important determinant of strain specific soil persistence.

Persistence

The natural habitat for EPN, the soil is a difficult environment for persistence of any organism considering its complexicity of physical, chemical and biological components. Nevertheless EPN have been isolated from soil throughout the world in ecosystems ranging from sub arctic to arid and temperate to tropical climates. It is assumed that along the course of evolution EPN just like other terrestrial organisms, adopted unique survival mechanisms to resist environmental extremes.
Nematodes like bacteria, fungi and plants can survive unfavourable environmental conditions in a dormant state which considerably prolongs their life span and enables them to withstand the rigours of a fluctuating regime. Dormancy can be separated into diapause and quiescence. Diapause is a state of arrested development and development does not recommence until specific requirements have been satisfied, even if suitable environmental conditions return. Quiescence is a facultative response, involving lowered metabolism, to unpredictable unfavourable conditions and is readily reversible when favourable conditions return, if the stress persists, some organisms can enter a state of cryptobiosis where is no measurable metabolism. Unfavourable environmental conditions include absence of water, extreme temperatures ,lack of oxygen and osmotic stress, the type of quiescens induced in an organism by these conditions are termed as anhydrobiosis, thermobiosis, cryptobiosis, anoxybiosis, and osmobiosis, respectively .While each of these factors may affect nematode survival, it is important to realize that in the soil these elements interact. Soil temperature is determined by factors that control heat transfer. Moist soil has greater conductance and increase in temperature than dry soil when exposed to the same heat source. Solute concentrations increase on drying of the soil. Therefore desiccation stress is commonly accompanied by pre exposure to osmotic stress .It is likely that physiological and biochemical mechanisms involved in tolerance to different stresses also interact.

Abstracts
1. Sweeney-J; Gesner-G; Bennett-R; Vrain-T. 1998. Effect of mulches on persistence of entomopathogenic nematodes (Steinernema spp.) and infection of Strobilomyia neanthracina (Diptera:Anthomyiidae) in field trials. AD: Canadian Forest Service, P. O. Box 4000, Fredericton, NB, E3B 5P7, Canada.
AB: The effect of mulches on the persistence of entomopathogenic nematodes, Steinernema spp., for infecting larvae of Strobilomyia neanthracina was tested in a series of field trials conducted in Picea in New Brunswick, Canada. Peat mulch increased the percentage of maggots infected with Steinernema bibionis strain 27, S. feltiae and S. carpocapsae. All strain, when maggots were placed on nematode-treated soil beneath the peat layer. However, when maggots were placed on the surface of the peat (to simulate larvae dropping) in a subsequent experiment, the percentage of maggots infected by S. feltiae strain 27 was less than half of that for maggots placed beneath the peat. Significantly fewer maggots were infected in peat than in either sand or potting soil in a laboratory experiment. Mean percentage of maggots infected by S. feltiae strain 27 over a 21-d period after application was not increased by retaining a natural vegetative cover, or by mulching with hay, wood-bark chips, or bark. Bark mulch, however, did reduce the rate of decline in percentage of infection over time. The numbers of S. feltiae infective juveniles present in the soil from 0 to 3 wk after application were estimated in 2 field trials by repeated baiting with greater wax moth larvae. Numbers of infective juveniles per gram of soil were positively related to percentage of cone maggots infected in 1 of 2 yr, but were not significantly affected by mulching. Mulching with hay, bark, or wood-bark resulted in significantly lower soil moisture potentials compared with non-mulched treatments. The results suggest that the mulches tested in this study would not maintain adequate efficacy or persistence of S. feltiae strain 27 for suppression of cone maggot over the 2- to 4-wk period of larval drop.

2. Ferguson-CS; Schroeder-PC; Shields-EJ. 1995. Vertical distribution, persistence and activity of entomopathogenic nematodes (Nematoda: Heterorhabditidae and Steinernematidae) in alfalfa snout beetle- (Coleoptera: Curculionidae) infested fields. Environmental-Entomology, 24:1, 149-158; 51 ref.
AB: The vertical movement, persistence and activity of 4 isolates of entomopathogenic nematodes (Heterorhabditis bacteriophora (Oswego), H. bacteriophora (NC), Steinernema carpocapsae (NY001) and an undescribed Steinernema species (NY008-2E)), were evaluated for 24 months at field locations in northern New York. Nematodes were released into 3 alfalfa [lucerne] fields naturally infested with Otiorhynchus ligustici. Each field differed in soil type and soil textural composition: silt loam sandy loam, and loamy sand. Nematodes were recovered from soil using trap insects (Galleria mellonella larvae) and their vertical distribution was monitored at 5-cm intervals to depths of 20 cm for Steinernema species and 35 cm for Heterorhabditis species. All nematodes persisted (no significant reduction in percentage of infection of G. mellonella) for 6 months after the initial application in all soil types. However, by the end of the 2nd growing season (17 months after application), all nematodes showed significant reductions in infection rates of G. mellonella except H. bacteriophora (Oswego) which showed high infection for 24 months. Nematode vertical movement was affected by soil type and varied by isolate. S. carpocapsae (NY001) and Steinernema sp. (NY008-2E) remained primarily in soil depths of < 15 cm, whereas both heterorhabditids dispersed to soil depths of 35 cm. Vertical movement of H. bacteriophora (Oswego) was greatest in loamy sand and vertical movement of Steinernema sp. (BY008-2F) was greatest in sand loam. The percentage infection of G. mellonella by H. bacteriophora (Oswego) and S. carpocapsae (NY001) was significantly correlated with rising soil temperatures in early spring. H. bacteriophora (Oswego) and S. carpocapsae (NY001) infected G. mellonella larvae in the field at soil temperatures between 15 and 18°C. Steinernema sp. (NY008-2E) infected G. mellonella larvae in the field at soil temperatures between 13 and 15°C.

3. Belair-G; Vincent-C; Chouinard-G.1994. Persistence of the entomopathogenic fungi Steinernema carpocapsae on apple. Resume-des-Recherches -Centre-de-Recherche-et-de-Developpement-en-Horticulture,-Saint-Jean-sur-Richelieu,-Quebec. 23: 3.
AB: The infectivity of Steinernema carpocapsae strain AII was assessed on apple leaves, flower clusters and twigs under field conditions. S. carpocapsae remained infective on apple leaves, flower clusters, and twigs up to 42 h after application. The infectivity of S. carpocapsae was higher on leaves than on flower clusters and twigs with an average of 90%, 70% and 36% insect mortality 36 h after application resp. On the flower clusters, no reduction in infectivity was monitored 36 h after the first application done at full bloom. On twigs on all application dates, a gradual reduction in nematode infectivity was recorded between 0 and 15 h. This was followed by a marked increase in infectivity 24 h after application.

4. Patel-MN; Wright-DJ. 1997. Fatty acid composition of neutral lipid energy reserves in infective juveniles of entomopathogenic nematodes. Comparative-Biochemistry-and-Physiology.-B,-Biochemistry-and-Molecular-Biology. 118:2, 341-348; 27 ref.
AB: The fatty acid composition of neutral lipids from infective juveniles (IJs) of Steinernema carpocapsae strain All, S. riobravis strain Biosys 355, S. feltiae strain UK76, and S. glaseri strain NC stored in distilled water at 25°C was determined. Newly emerged IJs of all 4 species had similar neutral lipid fatty acid profiles and of the 18 fatty acids identified, C18:1n-9 (43-49 mol %), C16:0 (18-23%), C18:2n-6 (8-14%) and C18:0 (4-8%) were the most abundant. Unsaturated fatty acids predominated, with about 50% being monoene and 14-22% polyene; the unsaturation index ranged from 91.6 in S. glaseri to 111.6 in S. carpocapsae. The fatty acid composition of the total lipid and the free fatty acid fraction mirrored that of the neutral lipids. During storage, the relative levels (%) of C16:0, C18:0, and C18:1n-9 in the neutral lipids declined significantly, suggesting they were preferentially utilized.

5. Wright-DJ; Grewal-PS; Stolinski-M. 1997. Relative importance of neutral lipids and glycogen as energy stores in dauer larvae of two entomopathogenic nematodes, Steinernema carpocapsae and Steinernema feltiae. Comparative-Biochemistry-and-Physiology. Biochemistry-and-Molecular-Biology. 118:2, 269-273; 17 ref.
AB: The infectivity of S. carpocapsae dauer larvae (infective juveniles) remained nearly constant up to 60 days of storage in water at 250C and then declined rapidly over the next 30 days. Few individuals remained infective after 120 days. Concurrent measurements showed that the mean neutral lipid content of individual S. carpocapsae declined to about 10% of initial levels after 60 days, and staining of individual nematodes with Oil Red O indicated that the population was almost homogeneous for low levels of neutral lipids. In contrast, the mean glycogen content of S. carpocapsae only declined significantly between 60 and 90 days of storage. These results showed that the decline in infectivity in S. carpocapsae is correlated primarily with the decline in glycogen reserves and it is suggested that glycogen is the key late energy store in aged infective juveniles. In contrast, S. feltiae dauer larva showed a much more gradual decline in infectivity over a 150- to 180-day storage period with a concurrent decline in neutral lipids, whereas glycogen levels declined up to 90 days of storage and then remained nearly constant. Thus, unlike S. carpocapsae, neutral lipids remain an important energy store in S. feltiae during storage, although glycogen also appears to be important, at least initially.

6. Simoes-N; Rosa-J. 1994. Survival of entomophilic nematodes in soil. Bulletin-OILB-SROP. 17:3, 77-80; 7 ref.
AB: The survival and pathogenicity of 2 nematode isolates from the Azores (Steinernema carpocapsae (Az20) and STEINERNEMA sp. (Az26)) were compared with those of S. glaseri in the field in the Azores and in the laboratory at 16°C. In sterile soil, the median survival time (ST50) was 2.9, 14.6 and 18.0 weeks, for S. carpocapsae, STEINERNEMA sp. and S. glaseri, resp. After 19 weeks, survival in sterile soil was 1, 32.9 and 29.8% for these species. The presence of the entomogenous fungus Metarhizium anisopliae in soil in the laboratory did not affect nematode survival. In both the laboratory and field, the pathogenicity of nematodes decreased significantly with time, the median pathogenicity time (PT50) for S. glaseri and S. carpocapsae being 8.7 and 7.0 weeks, resp. This paper was presented at the 4th general meeting of the IOBC/WPRS Working Group 'Insect pathogens and insect parasitic nematodes' held in Zurich, Switzerland, on 5-9 September 1993.

7. Nguyen-KB; Smart-GC Jr. 1990. Preliminary studies on survival of Steinernema scapterisci in soil. Proceedings -Soil-and-Crop-Science-Society-of-Florida, No.49, 230-233; 12 ref.
AB: The experiments reported here were conducted to acquire some information on the survival of S. scapterisci during summer and winter. All experiments were conducted by placing infective-stage juveniles of the nematodes in soil in containers which were either tightly sealed or sealed with a fine mesh cloth to prevent the nematodes from escaping. Containers were removed each week up to 14 weeks and the surviving juveniles recovered from the soil. Survival of the juveniles declined gradually over time, but 28% of those applied were recovered after 8 weeks when buried in tightly sealed containers of sterilized soil. When buried in cloth-sealed containers of sterilized soil, survival declined rapidly at the second week, but only gradually thereafter, with 16% recovered after 10 wk. At the end of each experiment, the juveniles recovered were infective. When infective juveniles were buried in tightly sealed containers of unsterilized soil, 29% survived at week 6 but survival declined rapidly thereafter with only 0.4% of the original number recovered at week 14. Those recovered at weeks 6 through 8 were infective; very few were recovered at weeks 9 through 14 and they were not tested for infectivity. Since in all tests 15 to 35% of the juveniles survived for 6 weeks and were infective. It is believed highly probable that the nematode will survive long enough when applied to the soil to encounter and infect a host.

8. Simoes-N; Rosa-J. 1994. Survival of entomophilic nematodes in soil. Bulletin-OILB-SROP. 17:3, 77-80; 7 ref.
AB: The survival and pathogenicity of 2 nematode isolates from the Azores (Steinernema carpocapsae (Az20) and Steinernema sp. (Az26)) were compared with those of S. glaseri in the field in the Azores and in the laboratory at 16°C. In sterile soil, the median survival time (ST50) was 2.9, 14.6 and 18.0 weeks, for S. carpocapsae, Steinernema sp. and S. glaseri, resp. After 19 weeks, survival in sterile soil was 1, 32.9 and 29.8% for these species. The presence of the entomogenous fungus Metarhizium anisopliae in soil in the laboratory did not affect nematode survival. In both the laboratory and field, the pathogenicity of nematodes decreased significantly with time, the median pathogenicity time (PT50) for S. glaseri and S. carpocapsae being 8.7 and 7.0 weeks, resp. This paper was presented at the 4th general meeting of the IOBC/WPRS Working Group 'Insect pathogens and insect parasitic nematodes' held in Zurich, Switzerland, on 5-9 September 1993.

9. Shamseldean-MM; Abd-Elgawad-MM. 1995. Survival and infectivity of entomopathogenic nematodes under environmental stress. Anzeiger-fur-Schadlingskunde,-Pflanzenschutz,-Umweltschutz. 68:2, 31-33; 17 ref.
AB: In 2 experiments in Egypt, infective-stage juveniles of 3 heterohabditid nematode isolates and Steinernema riobravis were placed in sterilized sandy soil in sealed containers buried in a maize field during summer. Containers were removed daily for 10 consequent days and surviving juveniles were recovered from the soil by the Baermann funnel method, then the remainder of soil was subjected to the Spodoptera littoralis baiting technique. Apparently, the low level of soil moisture (3%) and ceiling temperature levels (up to 42°C), as well as the faint electric conductivity (0.48 mmhos/cm) of the soil containing the nematodes resulted in a rapid decline in the number of nematodes recoverable from the soil over time by the Baermann method. The percentage recovery for the nematode isolates EAM8, EIS7 and EAS59 by the Baermann funnel was 35.6, 43.9 and 20.5 for the total nematodes recovered in the first trial, resp. The nematode numbers increased when the recovery time was extended from 24 to 48 h at 23°C. Insignificant differences were found between nematode numbers recovered daily in both trials. The average number of the recovered S. riobravis was more (P _0.05) than that of any other nematode isolate. Nematodes which were not recoverable by the Baermann technique were infective when bioassayed over the period of the 2 experiments. The infective juveniles which survived the stressed conditions are recommended to be subcultured by a selection regime in order to obtain an improved nematode strain.

10. Patel-MN; Perry-RN; Wright-DJ. 1997. Desiccation survival and water contents of entomopathogenic nematodes, Steinernema spp. (Rhabditida: Steinernematidae). International-Journal-for-Parasitology. 27:1, 61-70; 33 ref.
AB: The survival of exsheathed infective juveniles (IJs) of 4 Steinernema species, S. glaseri (NC),S. feltiae (UK76), S. carpocapsae (All) and S. riobravis (Biosys355), was assessed following fast and slow drying on glass slides and 1% (w/v) agarose, respectively. Freshly harvested and aged (75-day-old) IJs were desiccated on glass slides after removal of superficial water, at 0, 20, 40, 60 and 80% relative humidity (RH). Survival was assessed after rehydration with water and movement was used as the criterion for survival. Evidence for an intrinsic mechanism to control water loss and survive desiccation was found in freshly harvested S. carpocapsae IJs. At all humidities tested, S. carpocapsae had the greatest survival and the slowest rate of water loss. For example, at RH 80% the survival time for 50% (S50) of S. carpocapsae IJs was ca. 45 min compared with 5-20 min for the other species. Survival of aged IJs was markedly reduced in the case of S. carpocapsae and S. riobravis, and to a lesser extent in S. feltiae and S. glaseri. The 2nd-stage juvenile cuticle (sheath) was not important in aiding desiccation survival of S. carpocapsae and S. glaseri. Drying IJs slowly on 1% agarose at RH 80% greatly improved the survival of all 4 species, particularly S. glaseri and S. feltiae. The work is discussed in relation to possible mechanisms for survival of IJs during fast and slow drying.

11. Menti-H; Wright-DJ; Perry-RN. 1997. Desiccation survival of populations of the entomopathogenic nematodes Steinernema feltiae and Heterorhabditis megidis from Greece and the UK. Journal-of-Helminthology. 71:1, 41-46; 32 ref.
AB: The desiccation survival and ability to control water loss during drying of individual infective juveniles (IJs) of populations of Steinernema feltiae and Heterorhabditis megidis from Greece and the UK were examined at different relative humidities. Survival at all relative humidities was for minutes only and there was no evidence of enhanced survival of the populations of either species from Greece compared with those from the UK. The survival of individuals of both populations of H. megidis was superior to that of S. feltiae and the rate of drying of H. megidis was significantly slower. The sheath surrounding the IJs of H. megidis appears to be involved in slowing the rate of water loss of the enclosed nematodes.

12. Ogura-N; Nakashima-T. 1997. Cold tolerance and preconditioning of infective juveniles of Steinernema kushidai (Nematoda: Steinernematidae). Nematologica. 43:1, 107-115; 13 ref.
AB: Steinernema kushidai infective juveniles (IJs), that had been placed on to soil and stored at 5°C after recovery from artificial culture, did not survive well, with over 90% dying within 10 days. When the IJs were preconditioned at 10°C for over 8 days, a survival rate of over 50% was seen 100 days after storage at 5°C. This preconditioning at 10°C could also be carried out on IJs suspended in distilled water. Trehalose concentration in the IJs increased from almost undetectable levels to 1.4% of the dry weight when S. kushidai IJs in distilled water were preconditioned at 10°C for 20 days.

13. Grewal-PS; Gaugler-R; Wang-Yi; Wang-Y. 1996. Enhanced cold tolerance of the entomopathogenic nematode Steinernema feltiae through genetic selection. Annals-of-Applied-Biology. 129:2, 335-341; 32 ref.
AB: The SN strain of S. feltiae together with its bacterial symbiont, Xenorhabdus bovenii were selected for improved cold tolerance by repeated passage through the wax moth Galleria mellonella larvae at 15°C. Nematode virulence (total insect mortality and speed of kill) and establishment (initiation of nematode development following penetration) were evaluated after six (=12-24 generations) and 12 passages (= 24-36 generations). Cold selection enhanced nematode virulence at the cooler temperatures. Virulence measured as total insect-mortality at 8°C improved by 5.3- and 6.6-fold after six and 12 passages, resp. Only small improvements (1.2-1.5-fold) were observed in speed of kill. Nematode establishment improved at all temperatures after 12 passages; the highest increase of 9-fold was observed at 8°C. The results lend support to the hypotheses that functional traits along a continuously distributed environmental variable are genetically correlated and that the area under the fitness function is not always constant. Trade-offs in percentage mortality and speed of kill by the selected nematodes were observed at the warmer extreme after six passages of selection only. The implications of rapid changes in thermal sensitivity for economic mass-production of nematodes are discussed.

14. Menti-H; Wright-DJ; Perry-RN. 1997. Desiccation survival of populations of the entomopathogenic nematodes Steinernema feltiae and Heterorhabditis megidis from Greece and the UK. Journal-of-Helminthology. 71:1, 41-46; 32 ref.
The desiccation survival and ability to control water loss during drying of individual infective juveniles (IJs) of populations of Steinernema feltiae and Heterorhabditis megidis from Greece and the UK were examined at different relative humidities. Survival at all relative humidities was for minutes only and there was no evidence of enhanced survival of the populations of either species from Greece compared with those from the UK. The survival of individuals of both populations of H. megidis was superior to that of S. feltiae and the rate of drying of H. megidis was significantly slower. The sheath surrounding the IJs of H. megidis appears to be involved in slowing the rate of water loss of the enclosed nematodes.