Changes in Lipophorin Structure and Function during Phase Transition in Schistocerca gregaria (Forskal) (Orthoptera: Acrididae)

Abstract

The desert locust, Schistocerca gregaria is a major agricultural pest especially in the gregarious phase when it can cover an infestation area of approximate!~ 20% of the total land mass. The solitary phase on the other hand, occur in low densities with discontinuous distribution of no economic importance. The two locust phases differ in their anatont) behaviour, morphology, pigmentation, physiology and biochemistry. Swarming behaviour, a characteristic feature of the gregarious locusts, is associated with long distance flights of upto 5000 km. Locusts mainly use carbohydrates at the initiation of flight while, lipid is the main fuel in prolonged flight. Thus, lipid metabolism is of prime importance in the maintenance of long distance flights as in locust swarms. Due to the high flight activity in swarming (gregarious) locusts, in addition to the need for adequate lipid reserve, locusts require an efficient system for lipid mobilization, transport through the haemolymph and the ultimate utilization at the flight muscles. Although lipid metabolism plays a crucial role in prolonged flight, only limited studies have been carried out to determine how phase transition in locusts affects lipid metabolism. In order to understand how lipid metabolism is affected by phase transition in locusts, lipophorin, the major insect haemolymph lipoprotein involved in the transport of lipids from the fat body to the flight muscles, was chosen for the study. Differences between the two phases could in the long run be exploited in designing novel control strategies. Furthermore, it could also be important in distinguishing the locust phases either from field isolates or laboratory reared colonies. High- and low density- lipophorin (HDLp and LDLp) were isolated from both the solitary and gregarious S. gregaria by KBr density gradient ultracentrifugation (206,000 xg, 4° C, 4 h). HDLp isolated from the two phases had identical native molecular size of Mc 620,000 as determined by non-denaturing-PAGE. Analysis by SDS-PAGE showed the presence of similar subunits, apolipophorin-1 (apoLp-1, M, - 224,00) and apolipophorin-11 (apoLp-11, M, - 81,000). Both apoproteins were shown to be glycosylated with mannose rich oligosaccharide chains. LDLp isolated from both phases showed the presence of an additional apoprotein, apolipophorin-III (apoLp-III, M, - 20,000). The lipid percent content of the lipoproteins was determined gravimetrically in both phases. Further analysis of lipid moiety by gas chromatography indicated that the major lipid classes present in both HDLp and LDLp were phospholipids and diacylglycerides. Diacylglycerides in HDLp constituted 43.5 and 35.5% of the total lipids in gregarious and solitary locusts, respectively. On the other hand, it constituted 60 and 48 % in LDLp of the gregarious and solitary locusts, respectively. Immunological cross-reactivity was demonstrated between the lipoproteins isolated from the two phases as we11 as with antibodies raised against HDLp from Locusta migratoria. Haemolymph titres of lipophorin during the development of the locusts were estimated by single radial immunodiffusion. Starting from the third nymphal instar, there was a general increase in the levels as the insect developed, with the gregarious locusts having higher titres of the protein. Exceptions were observed during the fifth nymphal instar of the solitary locusts, when there was a large increase, which was higher than in the corresponding gregarious stage. ApoLp-111 was purified from LDLp by exploiting its solubility properties, stability to heat in addition to affinity chromatography on concanavalin-A-Sepharose. Analysis using SOS-PAGE showed that, apoLp-III isolated from the two phases had similar molecular size (Mr - 20,000). However, analysis on non-denaturing PAGE showed the presence of two isoforms in each case. Immunological cross-reactivity was demonstrated between apoLp-III isolated from both phases as well as with antibodies raised against L. migratoria apoLp-111. Estimation of the lipid reserve available in the locust fat body revealed that the gregarious locusts had a higher lipid reserve (79.02 ± 2.77%) as compared to the solitary ones (64. 75 ± 2.55 % ). Analysis of the fat body lipids by gas chromatography revealed that triacylglycerides was the major lipid type and constituted 83.9% and 73.85% of the total lipids in solitary and gregarious locusts, respectively. Lipid mobilization in response to administration of adipokinetic hormone (AKH), was demonstrated in both phases. A maximum response was observed 90 min after the hormone administration when the diacylglyceride levels reached a peak value. Furthermore, evidence of lipoprotein shift was obtained using gel permeation chromatography on an AcAn column. The response in gregarious locusts, led to the formation of a larger LDLp molecule than that formed in the solitary locusts. Increase in particle size was shown to be due to increase in diacylglyceride levels as well as association with more apoLp-III molecules. The response to a range of doses of AKH showed that the gregarious locusts were more sensitive to low doses below 2 pmol and the response was more tightly controlled than in solitary locusts. Thus, whereas gregarious locusts had a maximum response from 5 pmol, the solitary locusts peaked above 10 pmol. The ED50 was estimated to be 6.60 and 1.53 pmol for solitary and gregarious locusts, respectively. Activities of the fat body riacylglyceride lipase was studied using 14C-labelled triolein as the substrate tracer. The resting levels of the lipases was estimated to be 1.98 ± 0.24 and 1.95 ± 0.53 nmol/h/mg protein for solitary and gregarious locusts, respectively. Evidence of the activation of the lipase in response to AKH administration was deduced. Thus, administration of 2 pmol of AKH resulted in the elevation of the levels to 2.47 ± 0.39 and 2.30 ± 0.43 nmol/h/mg for gregarious and solitary locusts, respectively. Estimation of kinetic parameters showed that the solitary locusts K111 value was 18.75 compared to 46.67 µM for the gregarious locusts. The Vmnx \vas estimated to be 2.52 and 10.29 nmol/h/mg for solitary and gregarious locusts, respectively. The lipase from the gregarious locusts showed higher catalytic ability but lower affinity for the substrate. These properties of the enzyme may reflect a physiological adaptation arising from their metabolic requirements.