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

Abstract

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.

Fig 1

(Left) Metal rack holding the sample tubes, partly immersed in the ethanol bath of a refrigerated circulator. (Right) Sample chamber of the optical recrystallometer apparatus used for measuring RI activity.

Fig 2

Crystal diameters of nematode extracts (closed circles) and buffer controls (open circles) after annealing at −8°C for 30 min of splat frozen samples. Means ±SD, N = 10.

Fig 3

Splat frozen samples of S. feltiae extract (top row) and buffer control (25 mM Tris HCl, pH 8) (bottom row) after warming to – 8°C (Time 0 = left column) and after annealing at– 8°C for 30 min (right column). Scale bar = 100 μm.

Fig 4. The effect of temperature on the ice crystal size of the nematode extract after heating at various temperatures for 1 hr.

Means ± SD, N = 3.

Fig 5. The effect of time on optical transmittance (arbitrary units) in the recrystallometer.

Pure nematode extract (open circles), buffer (triangles), a 1:1 dilution of extract with buffer (closed circles) and a 1:3 dilution (squares) at annealing temperatures: -6°C (top graph), -7°C (middle graph) and -8°C (bottom graph). Means ±SD, N = 3.

Fig 6. The effect of dilution of S. feltiae extracts with tris buffer on crystal diameter (bars) and optical transmittance (arbitrary units) (line) after annealing for 30 min at – 8°C.

Error bars represent ±standard deviations, N = 10 for crystal diameter and 4 for optical transmittance.

Fig 7

Growth of a single Ice crystal in nematode extract (top) with clear hexagonal faceting, and a disc shaped crystal in Tris HCl (bottom) as seen on a Nanolitre osmometer. Scale bar = 100 μm.

Fig 8. Ice nucleation activity in S. feltiae in comparison with buffer control (25 mM Tris, pH 8).

Error bars are ±standard deviations, N = 24. Different small letters above the error bars represent that treatments are significantly different (P < 0.05).