C.H. Eisemann

Characterization of a Major Peritrophic Membrane Protein, Peritrophin-44, from the Larvae of Lucilia cuprina cDNA AND DEDUCED AMINO ACID SEQUENCES

The peritrophic membrane is a semi-permeable chitinous matrix lining the gut of most insects and is thought to have important roles in the maintenance of insect gut structure, facilitation of digestion, and protection from invasion by microrganisms and parasites. Proteins are integral components of this matrix, although the structures and functions of these proteins have not been characterized in any detail. The peritrophic membrane from the larvae of the fly Lucilia cuprina, the primary agent of cutaneous myiasis in sheep, was shown to contain six major integral peritrophic membrane proteins. Two of these proteins, a 44-kDa glycoprotein (peritrophin-44) and a 48-kDa protein (peritrophin-48) together represent >70% of the total mass of the integral peritrophic membrane proteins. Peritrophin-44 was purified and its complete amino acid sequence was determined by cloning and sequencing the DNA complementary to its mRNA. The deduced amino acid sequence codes for a protein of 356 amino acids containing an amino-terminal signal sequence followed by five similar but nonidentical domains, each of approximately 70 amino acids and characterized by a specific register of 6 cysteines. One of these domains was also present in the noncatalytic regions of chitinases from Brugia malayi, Manduca sexta, and Chelonus. Peritrophin-44 has a uniform distribution throughout the larval peritrophic membrane. Reverse transcriptase-polymerase chain reaction detected the expression of peritrophin-44 in all three larval instars but only trace levels in adult L. cuprina. The protein binds specifically to tri-N-acetyl chitotriose and reacetylated chitosan in vitro. It is concluded that the multiple cysteine-rich domains in peritrophin-44 are responsible for binding to chitin, the major constituent of peritrophic membrane. Peritrophin-44 probably has roles in the maintenance of peritrophic membrane structure and in the determination of the porosity of the peritrophic membrane. This report represents the first characterization of an insect peritrophic membrane protein.

Larvicidal activity of lectins on Lucilia cuprina: mechanism of action

Larvae of the blowfly Lucilia cuprina (Wied.) (Diptera: Calliphoridae) were grown in vitro on a serum‐free medium in the presence of a number of lectins. Lectins with specificities for β‐(1,4)‐N‐acetylglucosamine (wheat germ lectin) and α‐D‐mannopyranosyl and (α‐D‐glucopyranosyl residues (lentil lectin and Con A) caused strong concentration‐dependent inhibition of the growth of the larvae and substantial mortality. Wheat germ lectin had the strongest effects, showing 50% inhibition of larval growth at a concentration of 2 μM and 100% mortality at 25 μM. Other lectins with different sugar specificities had much less effect. The mechanism of the larvicidal action(s) of wheat germ lectin, lentil lectin and Con A was investigated. There were at least three effects of these lectins on L. cuprina larvae. First, these lectins bound to and reduced the permeability of the peritrophic membrane of the larvae. Second, they reduced ingestion of diet medium by larvae. Third, the lectins also bound to the apical membranes of larval gut epithelial cells although there were no obvious signs of damage to these cells. It is concluded that the combination of these effects probably results in the starvation of the larvae. The implications of these results in terms of possible control strategies for L. cuprina are discussed.

Lucilia cuprina: Inhibition of larval growth induced by immunization of host sheep with extracts of larval peritrophic membrane

A culture system has been established to produce gram amounts of peritrophic membrane from larvae of the sheep blowfly, Lucilia cuprina. Peritrophic membrane obtained from this culture has been used to immunize sheep. The immunization produced an immune response which resulted in the average weight of larvae on immunized sheep being only 50% of that of larvae grown on control sheep (P < 0.05). Fractionation of the components of the peritrophic membrane followed by immunization trials showed that the protective antigen fraction comprised material that could only be solubilized by harsh agents such as 4 M-urea. Even after solubilization by 4 M-urea, the protective antigens were able to produce a protective immune response which reduced growth of larvae on immunized sheep to 55% of larvae grown on control sheep (P < 0.05). This immune response which reduced growth of the larvae did not cause gross morphological damage to the larvae.

Isolation of a trypsin‐like serine protease gene family from the sheep blowfly Lucilia cuprina

Various protease inhibitors active against both trypsin‐ and chymotrypsin‐like serine proteases were used to characterize gut proteases from Lucilia cuprina by in vitro feeding assays. Significant larval growth retardation was observed on feeding first‐instar larvae with trypsin inhibitors, particularly soybean trypsin inhibitor. Feeding of chymostatin, a specific chymotrypsin inhibitor, resulted in no significant growth retardation. This information suggests that trypsin‐like serine proteases are probably the major gut digestive enzymes. A DNA fragment obtained by PCR which coded for part of a putative trypsin gene from L. cuprina was used to isolate a four‐member multigene family of trypsins. The full nucleotide sequence of one of the genes and partial sequence from the other three genes were determined. Transcription of at least one of the genes has been confirmed. All four of the genes appear to have arisen by two separate gene duplication events.

Chitin is only a minor component of the peritrophic matrix from larvae of Lucilia cuprina

The gut of most insects is lined with a peritrophic matrix that facilitates the digestive process and protects insects from invasion by micro-organisms and parasites. It is widely accepted that the matrix is composed of chitin, proteins and proteoglycans. Here we critically re-examine the chitin content of the typical type 2 peritrophic matrix from the larvae of the fly Lucilia cuprina using a range of techniques. Many of the histochemical and biochemical techniques indicate the presence of chitin, although they are often adversely influenced by the presence of highly glycosylated proteins, a principal component of the matrix. The alkali-stable fraction, which is used as an indicator of the maximum chitin content in a biological sample, is only 7.2% of the weight of the matrix. Larvae fed on the potent chitin synthase inhibitor polyoxin D or the chitin-binding agent Calcofluor White, showed strong concentration-dependent inhibition of larval weight and survival but no discernible effects on the matrix structure. A bacterial endochitinase fed to larvae had no effect on larval growth and no observable effect in vitro on the structure of isolated peritrophic matrix. RT-PCR did not detect a chitin synthase mRNA in cardia, the tissue from which PM originates. It is concluded that chitin is a minor structural component of the type 2 peritrophic matrix of this insect.

The peritrophic membrane: Its formation, structure, chemical composition and permeability in relation to vaccination against ectoparasitic arthropods

Peritrophic membrane (PM) lines the gut of many arthropods and other animals, and thus separates ingested food from the gut epithelium. Its main functions are connected with its partitioning of the gut lumen into regions between which the transfer of large macromolecules and other particles is limited by its permeability properties. In the context of vaccinating mammalian hosts against parasitic arthropods. PM may either restrict penetration of ingested immune effector components within the parasite, or serve as a target for immunological attack. The properties of PM that are relevant to these potential roles--its site and mode of formation, structure, chemical composition and permeability--are reviewed with reference to ectoparasitic insects. Recent experiments, in which sheep were vaccinated with extracts of PM from larvae of the sheep blowfly, Lucilia cuprina, are outlined. Antibodies ingested from vaccinated sheep slowed the growth of L. cuprina larvae. These antibodies bound specifically to the PM, reducing its permeability and thereby perhaps hampering utilization of food by larvae. The potential for vaccination against parasitic arthropods using antigens from their PMs is discussed.

Acquired resistance of sheep to larvae of Lucilia cuprina, assessed in vivo and in vitro.

The effect on subsequent larval survival of infesting sheep repeatedly with larvae of Lucilia cuprina was assayed in vivo and in vitro. One in vivo assay technique, in which implanted larvae were grown to third instar, indicated a significant reduction in larval survival; another in vivo technique, in which larvae were allowed to develop to second instar in small aluminium rings attached to the sheep, indicated no reduction in larval growth or survival. Larvae of Lucilia cuprina grown in vitro on media containing sera from previously infested sheep were significantly retarded in growth after 20 h compared with controls; no difference was detected when larvae were allowed to develop to pupation on two changes of the same media. No significant differences in survival of larvae either to 20 h or to pupation were obtained between the two treatments. ELISA antibody levels against crude soluble larval material were significantly higher for sera from infested sheep than for control sera, and the regression of antibody level on mean larval weight obtained after 20 h growth in vitro was significant. The immunoglobulin fraction isolated from sera of infested sheep significantly retarded larval growth when incorporated with normal serum in growth media. These results are consistent with an effect of specific anti-larval antibody produced by sheep in response to infestation.

CDNA AND DEDUCED AMINO ACID SEQUENCES OF A PERITROPHIC MEMBRANE GLYCOPROTEIN, 'PERITROPHIN-48', FROM THE LARVAE OF LUCILIA CUPRINA

The gut of most insects is lined with a semi-permeable peritrophic membrane (or peritrophic matrix) composed of chitin, proteoglycans and proteins. Despite the probable importance of the peritrophic membrane in facilitating the digestive process and protecting insects from invasion by micro-organisms and parasites, there has been little characterization of the specific components and their interactions within this acellular structure. Here we report the characterization of an integral peritrophic membrane glycoprotein, peritrophin-48, from the larvae of the fly Lucilia cuprina, a primary agent of cutaneous myiasis in sheep. Peritrophin-48 was purified from peritrophic membrane obtained by larval culture and its location within the peritrophic membrane determined by immuno-fluorescence and immuno-gold localizations. The cDNA coding for peritrophin-48 was cloned and sequenced. The deduced amino acid sequence codes for a protein of 375 amino acids containing an amino-terminal signal sequence followed by five similar, but non-identical domains, each approximately 65-70 amino acids in length and characterised by a specific register of six cysteines. The deduced amino acid sequence shows significant similarity to two other peritrophic membrane proteins, peritrophin-95 and peritrophin-44, from the same species. A reverse transcriptase-PCR approach indicated that there are several highly related peritrophin-48 genes expressed in each individual. Reverse transcriptase-PCR also demonstrated the expression of peritrophin-48 in all three larval instars and adults but not pupae or eggs. Peritrophin-48 was expressed only by the cardia and by the larval midgut. A simple structural model of a basic unit of a type 2 peritrophic membrane is presented.

The major excretory/secretory protease from Lucilia cuprina larvae is also a gut digestive protease

The larvae of the fly Lucilia cuprina excrete or secrete a chymotrypsia (LCTb) onto the skin of sheep to facilitate the establishment of the larval infestation. A combination of immunoblotting and RT-PCR approaches has established that this protease is also a gut digestive protease. LCTb is synthesized primarily in the cardia, a small highly specialized organ located at the anterior end of the midgut and by midgut cells. There is also some expression by the hindgut but no expression by salivary glands. Excretion of LCTb with waste products or regurgitation of the gut contents of the larvae may explain how this protease is transferred from the larval gut onto ovine skin. LCTb is first expressed in eggs and constitutively expressed throughout each larval instar, but is not expressed in pupae or adult flies. It is concluded that LCTb could be involved in the establishment of larvae on sheep skin as well as acting as a general gut digestive enzyme.

An estimate of the number of serine protease genes expressed in sheep blowfly larvae (Lucilia cuprina)

A large and diverse family of serine protease genes was identified in first‐instar larval cDNA of the sheep blowfly (Lucilia cuprina). This complex repertoire of genes was identified via a PCR approach using highly degenerate primers based on structurally conserved regions which surround the active site His and Ser residues found in all serine proteases. PCR products from entire first‐instar larval cDNA, or from third‐instar larval salivary glands or cardia, generated using a microscale RT‐PCR method, were cloned into a plas‐mid vector. Comparison of the restriction fragment patterns of PCR products generated from the three different sources suggests a highly diverse tissue‐specific pattern of serine protease expression in this organism. Detailed analysis of the restriction fragment patterns of sixty‐nine randomly selected clones from entire first‐instar larvae revealed forty‐nine different classes of PCR product. Maximum likelihood analysis of these data indicate that between 125 and 220 different serine protease genes are expressed in first‐instar larvae of L. cuprina. DNA sequence analysis of ten randomly‐selected clones, derived from the three tissue sources, indicated that all ten encoded serine protease gene fragments. A frequently occurring PCR product, generated from both first‐instar total cDNA and third‐instar cardia cDNA, showed 73% amino acid identity to a digestive protease expressed in Droso‐phila melanogaster larval gut cells.