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.

Gold labeling with wheat germ agglutinin and RNAse on osmicated tissue embedded in epoxy resin.

Tissue of an insect, Lucilia cuprina, fixed conventionally in buffered glutaraldehyde and osmium and embedded in epoxy resin (epon or epon/araldite), provided sections which could readily be labeled with RNAse/gold and wheat germ agglutinin (WGA)/gold. This method offers labeling of tissues with improved contrast and allows the retrospective application of RNAse and WGA labeling to conventionally prepared tissues, without recourse to oxidizing/etching agents.

Tick/host interactions for Ixodes holocyclus: role, effects, biosynthesis and nature of its toxic and allergenic oral secretions.

The Australian paralysis tick Ixodes holocyclus occurs along the eastern coast of Australia. Its interaction with a wide variety of hosts causes a serious toxicosis (tick paralysis) in domestic pets and livestock (occasionally in wildlife and humans) as well as hypersensitivity reactions in humans. Tick paralysis in animals is usually fatal in the absence of speedy antitoxin treatment and human hypersensitivity may result in life-threatening anaphylaxis. The protection of such hosts against toxic or allergic effects by vaccination or desensitisation respectively has been the objective of most of our recent research. The role, biosynthesis and nature of the paralysing toxin (holocyclotoxin) and of the allergens is gradually being elucidated. In this review, some emphasis has been placed on recent research on the interactions of humans with this tick and on the partial characterisation of the allergens.

Ultrastructure of the peritrophic membrane-secreting cells in the cardia of the blowfly, Lucilia cuprina.

Scanning and transmission electron microscopy are used to reveal the internal anatomy and ultrastructure of the cardia which is the source of the triple layered peritrophic membrane in the blowfly Lucilia cuprina. Within the cardia, rings of secretory cells (formation zones) and non-secretory tissue (valvula cardiaca) interlock to secrete and mould the layers of membrane. Formation zone cells have abundant rough endoplasmic reticulum, Golgi and secretory vesicles. A portion of midgut just posterior to the formation zone is covered by close-packed microvilli connected by septate-like junctions. The cuticle-lined valvula cardiaca is rich in smooth endoplasmic reticulum, glycogen and microtubules. The oesophageal cuticle is unusual in containing tubular structures. The ultrastructural features of the separate components of the cardia are discussed in terms of their secretory and non-secretory roles; modified midgut cells secrete chitin and protein whereas modified foregut tissue (valvula cardiaca) appears to be adapted to provide structural integrity (extensive junctions, microtubules), movement (muscles, possibly microtubules), a store of energy (glycogen deposits) and possibly a lipidic secretion (from smooth endoplasmic reticulum) to lubricate the passage of the membranes.

Ultrastructural localization of phenoloxidases in cuticle and haemopoietic tissue of the blowfly Lucilia cuprina.

The ultrastructural localization of two types of biochemically characterized phenol oxidase activity is described in the larva of the sheep blowfly, Lucilia cuprina. Cuticular tyrosinase activity (enzyme A) is seen in epicuticular filaments and procuticle. Procuticle activity can be detected only after a presumed process of activation takes place in damaged cuticle. By using either the dopamine reaction or inducing melanization by hot-water treatment, tyrosinase is readily shown in haemopoietic tissue which, in L. cuprina, occurs subdermally as well as being associated with the dorsal vessel. The adaptation of the diaminobenzidine technique, used to stain laccase in electrophoretic gels, to ultrastructural cytochemistry has made it possible to demonstrate enzymic activity probably due to laccase (enzyme B). The laccase activity is present in the inner epicuticle of late wandering third instar larvae (about to pupariate) but is not present in the epicuticle of younger larvae.

Ultrastructural changes in the cuticle of the sheep blowfly, Lucilia, induced by certain insecticides and biological inhibitors.

The ultrastructural effects on larval cuticle of Lucilia cuprina of two inhibitors of chitin synthesis, diflubenzuron and polyoxin D and an inhibitor of dihydrofolate reductase, aminopterin, are compared with those of the insecticide, cyromazine. Diflubenzuron and polyoxin D both prevent the formation of a normal lamellate appearance in procuticle and interfere with deposition of epicuticle. Aminopterin and cyromazine cause necrotic lesions in the cuticle which, in the case of cyromazine, are contiguous with invasive processes of epidermal cells. There is an accumulation of electron-dense granules in some epidermal cells in larvae poisoned with aminopterin or cyromazine. Aminopterin has a more drastic cytotoxic effect than cyromazine and it also interferes with the formation of epicuticle. The lesions produced by cyromazine treatment are not mimicked precisely by any of the other chemicals. However, there is closer accord between the effects of cyromazine and aminopterin than between cyromazine and the inhibitors of chitin formation.

Ultrastructural identification of neurohaemal sites in a tick: evidence that the dorsal complex may be a true endocrine gland.

A fine structural study has been made of the 'paraganglionic plates' associated with the perineurium and of the 'retrocerebral organ' associated with the periganglionic sheath of the tick Boophilus microplus; these structures have been postulated, from descriptions by light microscopy, to be tick neurohaemal organs. Neurosecretory terminals are observed frequently in the neural lamella/perineurial sheath, particularly in a dorso-lateral area which may correspond to the 'paraganglionic plates'. No evidence was found of a discrete peripheral neurohaemal organ such as the corpus cardiacum of insects. The 'retrocerebral organ' is comprised of periganglionic sheath cells, which appear to be glandular rather than neurohaemal, and peripheral ganglionic cells.

Improved toxin/antitoxin assays for studies on the Australian paralysis tick Ixodes holocyclus.

Sensitive biological assays of toxin/antitoxin potency have been developed to assist in research on characterization of salivary toxins of the Australian paralysis tick Ixodes holocyclus and on immunity to tick paralysis. The toxin assay utilizes suckling mice (4-5 g); a quantitative paralysis index is applied over a range of doses. The antitoxin assay is based on an in vitro/in vivo neutralization test which required a sensitive toxin assay and methods of standardization of toxin preparations. This assay permits the monitoring of blood antibody levels in animals during the course of development and loss of immunity and is assisting a study into the feasibility of producing an anti-paralysis vaccine. The method also allows standardization of commercial tick paralysis antiserum. The methods and applications are described and comparisons made with previous assays. Sample data are examined statistically by regression and variance analyses; parallelism of dosage-response lines is tested and relative toxicities (toxin) or potencies (antiserum) calculated.

Presence of T-bars, intramembranous particle arrays and exocytotic profiles in neuroendocrine terminals of an insect.

Definitive evidence is presented to show that arthropod neurohaemal terminals contain electron-dense T-bar structures with clustered microvesicles similar to those present in neuropilar and neuromuscular terminals. In terminal membranes of the locus corpus cardiacum, studied by freeze-fracture, intramembranous particle arrays, considered to correlate with the dense bars, are seen. However, there does not appear to be a spatial association between the arrays and the exocytotic profiles seen following exposure to stimulants for hormone release. The presence of the densities in both neuroendocrine and conventional terminals is discussed in the light of current theories for mechanisms of release of neurotransmitters and neurohormones in arthropods and vertebrates.

Perineurial and glial cells in the tick Boophilus microplus (Acarina: Ixodidae): freeze-fracture and tracer studies.

In the cattle tick Boophilus microplus, the cells of the perineurium are characterized by accumulations of glycogen which increase dramatically after feeding. Gap junctions couple both these perineurial cells which enshealth the C.N.S. and the underlying glial cells. No tight junctions have been found between perineurial cells and there is in consequence no blood-brain barrier. Using ionic lanthanum as a tracer the extensive gap junctions are shown to have no occluding effect and lanthanum penetrates through the perineurium and glial layers to the level of the axonal surfaces. By colloidal lanthanum impregnation and freeze-fracture studied, the gap junctions appear to be typical of arthropids in that their particles show a characteristic diameter (13 nm in freeze-fracture), are distributed relatively loosely within the junctional plaques and fracture onto the E face of the junctional membranes. Semi-ordered particle arrays are found on E face membranes of adjacent axons and glia which may represent axoglial junctions.

Norepinephrine as principal catecholamine in a specific neurone of an invertebrate (Boophilus microplus: Acarina).

Norepinephrine has been identified as the principal catecholamine in individual neurons of the synganglion of an arthropod, the cattle tick Boophilus microplus. This suggests that norepinephrine may have a hitherto unsuspected major physiological role in at least one group of invertebrates.

Prostaglandin in the saliva of the cattle tick Boophilus microplus.

Previous studies of saliva from engorged female cattle ticks revealed a component which induced contration of some isolated smooth muscles. Fractionation and further characterisation have shown that this substance is of the "slow-reacting" type, but that it is neither a bradykinin nor slow-reacting substance of anaphylaxis. The substance is deactivated by incubation with 15-hydroxprostaglandin dehydrogenase and its pharmacological properties also support its classification as a prostaglandin. A second pharmacologically-active component has now been found in the saliva but has not yet been characterised.