Research Article: Ice-Active Substances from the Infective Juveniles of the Freeze Tolerant Entomopathogenic Nematode, Steinernema feltiae

Date Published: May 26, 2016

Publisher: Public Library of Science

Author(s): Farman Ali, David A. Wharton, Neil T Wright.


Steinernema feltiae is a moderately freezing tolerant nematode, that can withstand intracellular ice formation. We investigated recrystallization inhibition, thermal hysteresis and ice nucleation activities in the infective juveniles of S. feltiae. Both the splat cooling assay and optical recrystallometry indicate the presence of ice active substances that inhibit recrystallization in the nematode extract. The substance is relatively heat stable and largely retains the recrystallization inhibition activity after heating. No thermal hysteresis activity was detected but the extract had a typical hexagonal crystal shape when grown from a single seed crystal and weak ice nucleation activity. An ice active substance is present in a low concentration, which may be involved in the freezing survival of this species by inhibiting ice recrystallization.

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Cold tolerant ectotherms have evolved a number of strategies to survive low temperatures [1, 2]. Short-term freezing survival, surviving the freezing event itself, is enhanced by a slow rate of freezing protecting animals from physical damage by slowing the rate of ice crystal growth [3]. Longer-term freezing survival of organisms however, also depends upon the production of a substance that inhibits recrystallization, and/or the production of low molecular weight compounds, e.g. trehalose [4, 5]. Many cold tolerant organisms produce proteins in response to reduced temperature that help them survive freezing. Those that interact with ice could be collectively named ice active proteins [6]. Some ice active proteins have the ability to bind to the ice surface thereby affecting the formation and stability of ice. They interact in different ways with ice, assisting the organism to survive sub-zero temperatures. They either inhibit the growth of ice (antifreeze proteins: [7, 8] or trigger ice formation (ice nucleating proteins: [9]. Antifreeze proteins are more common in freeze avoiding organisms, while ice nucleating proteins are often associated with freeze-tolerant organisms [10]. Antifreeze proteins inhibit the growth of ice by producing a thermal hysteresis. Thermal hysteresis is a non-colligative property resulting in freezing point depression, in the presence of an ice crystal, without changing the melting point. The difference in freezing and melting points is the amount of thermal hysteresis [11]. Ice nucleating proteins conversely, ensure ice formation at relatively high sub-zero temperature protecting the freeze-tolerant organism from the rapid ice formation that occurs at lower freezing temperatures. Ice nucleating agents serve as nuclei for ice crystal formation and can be either external to (exogenous ice nucleation) or present within the body (endogenous ice nucleation) of the organism [12].

Infective juveniles of S. feltiae showed a moderate level of RI activity in the splat freezing assay. This was indicated by smaller ice crystals after the annealing period in comparison to the buffer control. Heating the extract did not produce significant loss of RI suggesting the activity was relatively heat stable. The RI activity of P. davidi IAP is also relatively heat stable [6] and so is that of several of the plant AFPs [22]. The level of RI was moderate, with an increase in crystal size during annealing and a reduction in RI after 1:3 dilution, almost to that of a buffer control. Smith et al [16] compared the RI activity of six nematode species including an entomopathogenic nematode, S. carpocapsae. The annealing period and the concentration used (20 min, 18 mg/ml) were different than that in the present study (30 min, 25.3 mg/ml), so the results cannot be directly compared. However, despite the lower concentration and shorter annealing time, the crystal size was smaller in two of the six species (S. carpocapsae and P. davidi) than that of the S. feltiae extract shown here. This places S. feltiae in a moderate RI category. However, a moderate level of RI may be of importance for this moderately freeze tolerant species which survives intracellular freezing to -3°C [23]. Ramløv et al [19] suggest that for a freeze tolerant animal, RI may be important to survive the freezing stress. It plays a role in inhibiting the growth of ice crystals and/or in controlling the size, shape and location of ice crystals after their formation [6, 24]. The moderate RI activity of S. feltiae may play a role in the size and shape of ice crystals, as indicated by their appearance in freeze-substituted specimens [23]. This protects the nematode from damage and enables it to survive intracellular freezing to -3°C but not to the same extent as P. davidi, which has strong RI activity and survives to much lower temperatures [14]. However, there is not a direct correspondence between RI activity and nematode survival [14], suggesting that other factors are also important.