Biophysical properties of the surface of desiccation-tolerant mutants and parental strain of the entomopathogenic nematode Heterorhabditis megidis (strain UK211).

Entomopathogenic nematodes (EPN) are useful biological control agents of insect pests. However, the infective juvenile (IJ) stage which is the only stage to occur outside the host is susceptible to environmental extremes such as desiccation. We have isolated desiccation-tolerant strains of the EPN Heterorhabditis megidis. In this paper we describe the surface properties of these desiccation-tolerant mutants. Heterorhabditid IJs retain the sheath of the previous larval stage. The mutant lines possess alterations in the surface properties of the sheath. Differences were observed in fluorescent lipid analogue insertion into the surface of the sheath. Furthermore, cationized ferritin-binding studies demonstrated that the mutant lines possessed an increase in net negative surface charge. Removal of the surface layer of the sheath resulted in the loss of the mutant phenotype and in a reduction in the desiccation tolerance of the parental strain. Therefore, the negatively charged 'surface coat' appears to play an important role in the desiccation tolerance of Heterorhabditis species.

Influence of culture method on Steinernema glaseri lipids.

Entomopathogenic nematodes can be mass produced in artificial media for use as biological insecticides. Nematode in vitro media have been primarily developed on the basis of yield without fully considering nematode nutritional requirements. We investigated the quality and quantity of lipids in the entomopathogenic nematode Steinernema glaseri when grown in vivo in Popillia japonica (a natural host), Galleria mellonella (a factitious host), and in solid and liquid media. Nematode yield (infective juveniles per mg dry organic material) was 4 times higher in the in vivo compared with the in vitro cultures. Nematodes produced in vivo using P. japonica accumulated a significantly higher amount of lipids compared with nematodes grown using G. mellonella or in vitro solid and liquid methods, respectively. Fractionation of S. glaseri total lipids revealed that nematodes produced using P. japonica accumulated significantly higher phospholipids and sterols compared with other methods. C:18 fatty acids were the predominant class of lipids in S. glaseri irrespective of production method. In vivo-produced nematodes had oleic 18:1 acid as the major fatty acid, whereas in vitro-produced S. glaseri had a mixture of oleic 18:1 and linoleic 18:2 acids as the predominant fatty acids. We conclude that the lipid composition of entomopathogenic nematode is host or medium dependent. We suggest that adjusting the in vivo medium by addition of components similar to a natural host nutritional composition should improve nematode production.

Glycogen: its importance in the infectivity of aged juveniles of Steinernema carpocapsae.

Infective juveniles (IJs) of Steinernema carpocapsae (All) are able to remain relatively highly infective even when they have almost exhausted their neutral lipid reserves. This is not seen in other steinernematid species so we proposed that carbohydrate may be important for infectivity in aging IJs of S. carpocapsae. The present study investigated glycogen utilization in IJs of 4 entomopathogenic nematodes, S. carpocapsae, S. riobravis (Biosys 355), S. feltiae (UK76) and S. glaseri (NC), stored in distilled water at 25 degrees C. The 4 species had appreciable amounts of glycogen; from ca. 8% dry weight in S. riobravis to ca. 18% in S. glaseri. Infective juveniles of S. carpocapsae and S. riobravis survived for 120-135 days and utilized ca. 90% of their glycogen reserve at an almost constant rate during a 112-day storage period. Steinernema feltiae and S. glaseri lived for much longer (> 450 days) and during a 250-day storage period their glycogen content decreased by 27 and 40%, respectively. In contrast to the other 3 species, the rate of lipid decline preceded that of glycogen in S. carpocapsae. The rate of glycogen decline in S. carpocapsae IJs incubated with the glycolytic inhibitor, iodoacetamide (10(-4) M) was significantly reduced (P < 0.05) compared with untreated nematodes, and the infectivity of inhibitor-treated aged (> 80 days) IJs was reduced compared with controls. Incubating aged (80-day) IJs of S. carpocapsae (mean neutral lipid content ca. 10% of initial level) with 10(-4) M iodoacetamide for 24 h significantly reduced (P < 0.05) their infectivity compared with freshly harvested inhibitor-treated IJs and untreated controls. Following an 11-day recovery period, the infectivity of inhibitor-treated aged IJs recovered significantly (P < 0.05). The evidence suggests that glycogen is an important source of energy for maintaining infectivity in aged IJs of S. carpocapsae.

Desiccation survival and water contents of entomopathogenic nematodes, Steinernema spp. (Rhabditida:Steinernematidae).

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 (r.h.). 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 r.h.s tested, S. carpocapsae had the greatest survival and the slowest rate of water loss. For example, at 80% r.h. 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 80% r.h. 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.