Proteases released by Lucilia cuprina during egg hatch

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Abstract

Products released by Luclia cuprina (sheep blowfly) during egg hatch and early larval development were found to contain a variable number of proteases, probably reflecting their different functions during these developmental phases. Moreover, a number of the developmentally regulated proteases produced only during egg hatch appeared to be strongly egg shell associated, indicating the egg shell proteins to be their specific substrates. Several proteases were tested for their ability to enhance the rate of egg hatch. The commercial proteases chymotrypsin and trypsin were able to significantly enhance (p≤0.01) egg hatch by 36% and 44%, respectively, when compared to an untreated control. Interestingly, fluids containing the egg shell associated proteases were able to significantly (p≤0.001) enhance L. cuprina egg hatch by up to 70%. This hatch enhancement activity could be significantly (p≤0.001) reversed by the addition of Phenylmethylsulphonyl fluoride (PMSF). Fractionation of the egg-associated proteases by gelatin affinity chromatography suggested that gelatin-binding molecules were responsible for the majority of the egg hatch enhancement activity in this preparation.

Introduction

The hatching of parasite eggs is an important process which represents a critical phase in the development of parasitic infections. Due to the importance of this process for parasite survival, the egg hatching of many gastrointestinal parasites has been studied in great detail, indicating that secreted enzymes often facilitate successful endoparasite egg hatch (Rogers and Brooks, 1977; Holmes and Fairweather, 1982; Xu and Dresden, 1986; Bock, 1989; Arnold et al., 1993). A number of different enzymes have been identified in the hatching fluids of endoparasites including proteases, lipases, chitinases, α-glucosidases, β-glycosidases and leucine aminopeptidases (Rogers, 1958; Edmonds, 1966; Barrett, 1976; Hinck and Ivey, 1976; Rogers and Brooks, 1977; Xu and Dresden, 1986; Bock, 1989; Arnold et al., 1993). In contrast, the hatching of ectoparasites, or other insects to which most ectoparasites are closely related, has not been studied in the same detail. There are a few examples of insect egg hatching involving the secretion of enzymes (Slifer, 1937; Jones, 1956; Praw, 1961; Novâk and Zambre, 1974), but most research into insect egg hatching has focussed on the mechanical aspects of the hatching process (Balfour-Browne, 1913; Puri, 1925; Sikes and Wigglesworth, 1931; Wigglesworth, 1932; Jackson, 1948; Rodriguez et al., 1992).

Studies of the hatching process of L. cuprina eggs have suggested that egg hatch may be facilitated by a combination of both physical/mechanical and biochemical processes (Young, Personal Communication). In addition, previous studies have found that insect eggs are composed predominantly of protein (Margaritis, 1985), consistent with a role for proteases in the egg hatching process. Earlier studies of L. cuprina excretory-secretory (ES) products involved in wound initiation (Young et al., 1996) have identified a number of proteases, the transient detection of which implied that they were possibly developmentally regulated, being required during development to perform a specific function. Studies were therefore undertaken to identify developmental proteases produced during early L. cuprina development, particularly during egg hatch.

Section snippets

Collection of hatching fluids

L. cuprina eggs were supplied by Dr. M. Sandeman, School of Agriculture, La Trobe University and Dr. J. McKenzie, Department of Genetics, the University of Melbourne. The collection of samples for analysis is schematically represented in Fig. 1. After being laid onto sheep liver, L. cuprina eggs were removed from the liver and moistened with ddH2O to separate the egg masses. Eggs were washed 3 times with approximately 10 times their volume of ddH2O and placed in a sterilising solution of 0.25%

Gelatin substrate SDS-PAGE analysis of protease collected during the hatching of L. cuprina eggs

Gelatin substrate SDS-PAGE analysis of proteases collected during the hatching of L. cuprina eggs

Gelatin substrate SDS-PAGE detected a variety of proteases associated with hatching L. cuprina eggs (Fig. 2). The hatch fluid contained at least eleven protease species that differed in apparent molecular weight; the most active proteases had sizes of approximately 250 kDa and below 50 kDa (Fig. 2, lane 1, arrowed). To determine the presence of developmental proteases specific for hatching, which may

Discussion

Many different proteases were released during the hatching of L. cuprina eggs and a number of these appeared to be specifically associated with the egg shell. These proteases were only released from the hatched egg shells after either extensive washing or following homogenisation in the highly denaturing SDS-PAGE sample buffer. The close association of these proteases with the egg indicated a high affinity for the egg shell, consistent with the proposal that the egg shell is the specific

Acknowledgements

Anna R. Young is the recipient of an Australian Postgraduate Research Award (Industry).

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