Abstract
Entomopathogenic nematodes (EPN), despite their great biological control potential, cannot currently compete with conventional chemical pesticides. Among other reasons, their limited shelf-life is the major impediment; which makes their production costs high, and thus limits their application to use against selective insect pests of high value crops only. Attempts to base a storage technology on partial desiccation and anhydrobiosis have met with limited success. This thesis investigates the cold tolerance abilities of two EPN species, Steinernema feltiae and Heterorhabditis bacteriophora, and the underlying mechanisms involved in the hope that a storage technology based on freezing may be possible.
S. feltiae and H. bacteriophora have modest levels of cold tolerance; with 50% survival temperatures (S50) of –12.64 °C and −11.78 °C respectively. This level of survival is achieved when freezing of the nematodes is nucleated by ice and they are left overnight at −1 °C after cooling at 0.5 °C min-1. Acclimation (two weeks at 5 °C) significantly enhances the survival of S. feltiae (S50, −13.16 °C), but not of H. bacteriophora. This effect is in addition to that of the overnight freezing effect (at −1 °C). There was no response to rapid cold-hardening or cold shock under the conditions tested.
Infective juveniles of S. feltiae avoid inoculative freezing at −1 °C as observed on a microscope cold stage, suggesting the occurrence of partial cryoprotective dehydration. However, at temperatures −2 °C and below, the nematodes freeze completely confirming that the species survives predominantly through a freezing tolerance strategy.
The pattern of ice crystal formation and location was further visualised using freeze substitution and transmission electron microscopy. It was found that S. feltiae can survive intracellular ice formation, providing the second example of intracellular freezing survival amongst animals (after the Antarctic nematode, Panagrolaimus davidi). The pattern of ice crystal formation was not as controlled as in P. davidi. Both small and large ice crystals were found in individual nematodes but small ice crystals were more common in nematodes frozen at high sub-zero temperatures than at lower temperatures. Snap-frozen nematodes in liquid nitrogen had only small ice crystals, but did not survive.
Low molecular weight cryoprotectants were involved in the freezing tolerance of S. feltiae. The nematodes accumulate large quantities of trehalose and glycerol in response to cold acclimation (2 weeks at 5 °C) and the freezing process per se, which appear to protect the nematodes from freezing injury by reducing the formation of ice and replacing the lost water.
A high molecular weight cryoprotectant was found to be inhibiting recrystallization in the frozen nematodes. This presumed protein is heat-stable, but has no thermal hysteresis or ice nucleation activity, and is thus termed a recrystallization inhibiting protein. The recrystallization inhibition activity was moderate, but could enable a freeze tolerant animal to control the size, shape and location of ice crystals after freezing.
The modest freeze tolerant abilities of these nematodes in part reflect their response to the environment in which they live in. A survey of cold tolerant EPN in Otago revealed that the distribution of EPN was limited to low and medium altitude sites (<700 m a.s.l). Two species, S. feltiae and S. kraussei were recovered; the latter being the first record of this species from the Southern Hemisphere. This study also reports the isolation of a virulent strain of a nematophagous/entomopathogenic fungus (Pochonia bulbillosa) from the Rock and Pillar Range of Otago, New Zealand.
This study suggests that the natural capacity of EPN for freezing tolerance could be exploited as a first step towards developing a method for their long-term storage.