Bioenergetics in a parasitic nematode, Steinernema carpocapsae, monitored in vivo by flow NMR spectroscopy

Parasitol Res (1991) 77:86-90

Parasitology Research 9 Springer-Verlag 1991

Bioenergetics in a parasitic nematode, Steinernema carpocapsae, monitored in vivo by flow NMR spectroscopy S.N. Thompson, E.G. Platzer, and R.W.-K. Lee Analytical Chemistry Instrumentation Facility and Departments of Entomology, Nematology and Chemistry, University of California, Riverside, CA 92521, USA Accepted August 26, 1990 Biochemical research on helminth parasites is aimed at characterizing their metabolic pathways, particularly those involved in energy production, in the hope of identifying parasite-specific metabolic targets for development of new and more effective chemotherapeutic drugs (Bryant and Behm 1989). Recently, attention has focused on the developmental transitions between aerobic and anaerobic metabolism that often occur between the free-living infective and parasitic stages (Barrett 1986). Through unique application of a flow system in in vivo analysis by nuclear magnetic resonance spectroscopy (NMR), whereby the organisms themselves were continuously recirculated through the spectrometer, we observed aerobic-anaerobic transitions in a single developmental stage of the nematode Steinernema carpocapsae, parasitic in insects. High levels of phosphoarginine were observed under aerobic conditions and its relative level decreased sharply during the transition to anaerobic metabolism. N M R has previously been applied to in vivo analysis of whole animals including several helminths (O'Sullivan 1989). A problem plaguing many in vivo N M R investigations, however, is the protracted time required to generate meaningful data and the difficulty in maintaining animals under controlled conditions during analysis. This is due to the relative insensitivity of N M R compared with more traditional analytical methods (Gadian 1982). In addition, the small size of many invertebrates causes extreme difficulty in retaining and perfusing animals in the traditional manner while in the spectrometer. We describe below the successful application of in vivo flow N M R , a method that both maintains animals under aerobic conditions and reduces spectral generation time in pulsed Fourier transform N M R experiments. By flowing the animals through the spectrometer, saturation effects resulting from incomplete relaxation of nuclei were avoided and short interpulse delays could be used. In vivo spectra of relatively small tissue mass were generated within several minutes. Moreover, in contrast to many in vivo N M R perfusion studies that employ Offprint requests to: S.N. Thompson

less than optimal pulse width to reduce spectral generation time, the flow method enabled use of 90 ~ pulses for optimizing sensitivity. Previous N M R studies with flowing aqueous and organic solutions have demonstrated that signal enhancement by continuous efflux of saturated nuclei can be as much as two fold for nuclei with spin lattice relaxation times (T1) of 5 to 10 s and 10-fold for nuclei with longer Tls (Laude et al. 1984, 1985). Third-stage larvae of S. carpocapsae, kindly donated by Dr. R. Georgis and Biosys, Inc, Palo Alto, CA, were suspended in a 0.05M NaC1 containing 0.01 M 4-(2-hydroxyethyl)- 1-piperazine-ethanesulphonic acid (HEPES) buffer, pH 7. Each experiment utilized ~ 2 • 107 worms (dry weight 1.8 g) in 50 ml medium at 24~ C. The nematode suspension was pumped continuously through 1/16 id vinyl tubing into a 12-ram N M R tube and recirculated into a small conical reservoir with the aid of a peristaltic pump. The N M R tube was positioned in the probe of a Nicolet 300 superconducting spectrometer interfaced with a Nicolet 1280 computer and operated in the pulsed Fourier transform mode. The nematodes entered the probe through the bottom of the magnet and passed through a 2.5 cm premagnetization region in the probe before entering the radiofrequency coil. In the present studies a flow rate of 60 ml. min- ~ was used and nematodes passed through the probe at a velocity of approximately 1.00 cm/s. The nematode mass contained within the probe's radiofrequency coil volume at any time was approximately 70 mg dry weight. The nematode suspension was gassed in the reservoir with 5% CO2 in 95% O2 to establish aerobic conditions. Calculations based on results of published oxygen uptake studies (Burman and Pye 1980) indicate that the oxygen tension in the suspension was sufficient to maintain the worm mass under aerobic conditions while flowing through the spectrometer. To examine nematodes under hypoxic conditions the suspension was gassed with 5% CO2 in 95% N2. Following each experiment, the nematodes were examined under the microscope. Based on mobility, 90 to 95% remained viable. In vivo 31p N M R spectra were generated at

S.N. Thompson et al. : Flow NMR studies on a parasitic nematode 121.5 MHz. A single 90 ~ (50 gs) pulse and 0.25 s acquisition time were employed. The delay time between acquisition and the next pulse had a major impact on the relative intensity of spectral components, particularly those with long Tls. At a flow rate of 60 ml/min, a total interpulse delay of at least 3 s, that is approximately 2.75 s following acquisition, was necessary to obtain maximal intensity of all spectral peaks. Intensity corrections were necessary when shorter interpulse delays were used and standard intensity curves were developed from spectra generated with total interpulse delays of 0.25 to 15s. The in vivo 31p N M R spectrum of S. carpocapsae was composed of seven spectral peaks (Fig. 1 A). Assignments were made by comparing chemical shifts with those reported in the literature (Gadian et al. 1979), comparison of chemical shifts with standards in pure solutions, and/or spiking tissue extracts (see below). The presence of phosphoarginine in tissue extracts was further verified by addition of arginine kinase. Under aerobic conditions phosphoarginine was a major phosphorus component (Fig. 1 A), but the phosphagen was absent in nematodes maintained under hypoxic conditions (Fig. 1 B). The levels of phosphoarginine and ATP were repeatedly decreased upon short-term exposure of nematodes to hypoxia (Fig. 2). Based on the chemical shift of inorganic phosphate (Pi) in the in vivo spectrum, only minor decreases in pH,