Antibody-mediated inhibition of the growth of larvae from an insect causing cutaneous myiasis in a mammalian host

Abstract

Many insects feed on blood or tissue from mammalian hosts. One potential strategy for the control of these insects is to vaccinate the host with antigens derived from the insect. The larvae of the fly Lucilia cuprina feed on ovine tissue and tissue fluids causing a cutaneous myiasis associated with considerable host morbidity and mortality. A candidate vaccine antigen, peritrophin 95, was purified from the peritrophic membrane, which lines the gut of these larvae. Serum from sheep vaccinated with peritrophin 95 inhibited growth of first-instar L. cuprina larvae that fed on this serum. Growth inhibition was probably caused by antibody-mediated blockage of the normally semipermeable peritrophic membrane and the subsequent development of an impervious layer of undefined composition on the gut lumen side of the peritrophic membrane that restricted access of nutrients to the larvae. The amino acid sequence of peritrophin 95 was determined by cloning the DNA complementary to its mRNA. The deduced amino acid sequence codes for a secreted protein containing a distinct Cys-rich domain of 317 amino acids followed by a mucin-like domain of 139 amino acids. The Cys-rich domain may be involved in binding chitin. This report describes a novel immunological strategy for the potential control of L. cuprina larvae that may have general application to the control of other insect pests.

Figure 1

SDS/PAGE of purified peritrophin 95. (a) Silver-stained SDS/PAGE of peritrophin 95 (reduced). Lanes: 1, standards; 2, peritrophin 95 (2 μg). (b) Biotinylated lectin blot of purified peritrophin 95 (5 μg, lanes 2 and 3). Lanes: 1, standards; 2, biotinylated wheat germ lectin; 3, biotinylated wheat germ lectin after incubation of the filter with 0.3 M GlcNAc.

Figure 2

Anti-larval effects of serum from sheep vaccinated with peritrophin 95. The effects of anti-peritrophin 95 serum from eight sheep on the mean weight (a) and mean survival (b) of L. cuprina larvae were measured (shaded histograms) by using an in vitro feeding bioassay. The unshaded histograms refer to corresponding results for a control group of four sheep. Vertical bars on histograms indicate 1 SD. Numbers above the control and peritrophin 95 groups represent group means ± 1 SD. The mean weight of larvae feeding on the serum from sheep vaccinated with peritrophin 95 was significantly different from the control group (P < 0.001).

Figure 3

Enhanced larval growth inhibition using serum enriched with Ig isolated from a sheep vaccinated with peritrophin 95. Larvae were allowed to feed on the Ig-enriched serum for 20 h in an in vitro feeding bioassay after which mean weight (a) and mean survival (b) were measured. Controls, Ig from control sheep supplemented into NSS at 1-, 2-, and 4-fold enrichments (unshaded); Ig, (anti-peritrophin 95) immune Ig supplemented into NSS at 1- (1× Ig), 2- (2× Ig), and 4- (4× Ig) fold enrichments (shaded); serum, original unmodified anti-peritrophin 95 serum. Error bars denote 1 SD.

Figure 4

Reduced permeability of the PM. Larvae were fed on NSS enriched 4-fold with Ig from control serum (a) or anti-peritrophin 95 serum (b). Both Ig-enriched sera contained 6-nm colloidal gold particles. The larval PM was then examined by transmission electron microscopy. PM, peritrophic membrane; MV, microvilli of gut digestive epithelial cells; ENPS, endoperitrophic space; ECPS, ectoperitrophic space; B, bacterium; L, antibody-induced layer on gut lumen side of PM. Colloidal gold particles are indicated by arrows.

Figure 5

Nucleotide and deduced amino acid sequences of peritrophin 95 determined from cDNA. Single underlining denotes peptides determined by amino acid sequencing. Cys residues are circled. A potential polyadenylylation signal sequence is underlined twice. The position of the amino terminus of the mature protein is indicated by an arrow and the amino-terminal signal sequence is underlined with a broken line. Potential N-linked glycosylation sites are boxed. The 5′ and 3′ noncoding regions are represented by lowercase type.

Figure 6

Domain structure of peritrophin 95 and similarities with other proteins. (a) Alignment of five related six Cys subdomains in peritrophin 95 with similar domains from other proteins. PM95 I–V, the five consecutive peritrophin 95 six Cys subdomains; PM44 I–V, the five consecutive six Cys subdomains from the L. cuprina PM protein peritrophin 44; Ms, Ch, Av, Ov, and Bm are single six Cys carboxyl-terminal domains of chitinases from Manduca sexta, Chelonus sp., Acanthocheilonema viteae, Onchocera volvulus, and Brugia malayi, respectively. Cysteines are boxed, potential N-linked glycosylation sites are underlined, and the positions of semiconserved aromatic amino acids are indicated by an asterisk. The symbol > represents the carboxyl-terminal end of a protein. (b) Type I and type II repeated sequences in the carboxyl-terminal 139 amino acid of peritrophin 95. Prolines in the type I repeats are boxed. (c) Sequence similarity between T. brucei insect-stage procyclin (parpAa) and a region in peritrophin 95 (PM95).