Uptake and persistence of ingested antibody in the mosquito Anopheles stephensi.

A sensitive ELISA was developed to monitor the persistence of a specific antibody, rabbit anti-BSA, in the bloodmeal, haemolymph and tissues of the mosquito Anopheles stephensi Liston. Different concentrations of anti-BSA were fed to female mosquitoes in sheep blood, via a membrane-feeder, and it was found that antibody persisted in the gut as the bloodmeal was digested: concentrations present at 24 h were directly related to those fed. Homogenates of mosquito bodies, from which the intact guts had been removed, were always antibody-positive up to 9 days post-feeding, indicating that undigested antibody had passed through the gut wall into the haemocoele. Haemolymph was extracted from mosquitoes at different times post-feeding, using a microcapillary and manipulator, and antibody was detected in several of the assays. The level of antibodies in the haemolymph 24 h post-feeding was less than half of the level in mosquito heads, indicating removal of antibodies from the haemolymph, perhaps by binding onto haemocoelic tissues. The relevance of these results to the ingestion, survival and fate of antibody against malaria sporozoites is discussed.

The role of galactosyl-binding lectin in the cellular immune response of the cockroach Periplaneta americana (Dictyoptera).

Agglutinating activity of the major serum lectin of Periplaneta americana against mammalian erythrocytes has been found previously to be inhibited most effectively by galactose and glycoproteins rich in non-reducing terminal galactose, such as porcine stomach mucin (PSM) and desialylated fetuin and bovine submaxillary mucin. Antibody raised against the purified lectin was found, by immunofluorescence, to bind to the surface of washed fixed haemocytes (the cells responsible for cellular immunity in insects). The experiments described here were carried out in order to investigate whether or not the lectin plays any role in mediating the haemocytic response, by measuring the in vivo cellular response to galactose- or sialic acid-rich glycoproteins, either in solution or conjugated to Sepharose beads. Comparison of the ability of injected solutions to stimulate haemocytic aggregates (nodules) showed that PSM, whether native, desialylated or pronase-digested to produce small galactose-containing fragments, stimulated large numbers of nodules, in contrast to either sialic acid-rich glycoproteins such as bovine submaxillary mucin (BSM) and fetuin, or the protein bovine serum albumen (BSA). Multicellular capsules formed around Sepharose conjugated to galactose-rich molecules such as PSM, asialo-PSM, asialo-fetuin or asialo-BSM were highly significantly thicker than those formed around control untreated beads, whereas capsules around BSA, fetuin or BSM conjugates were significantly thinner. It is unlikely that the different results for sialylated and desialylated molecules are due merely to a non-specific charge effect, since the response due to charge alone is directly opposite to that found when specific carbohydrate groups are considered. The results tentatively support the idea that soluble and/or cell-associated lectins may be involved in immuno-recognition in insects.

Immune mechanisms in insects.

Understanding immune evasion by parasites in their insect vectors requires some understanding of the insect immune system. Until fairly recently, technical difficulties in handling cells and plasma hampered laboratory investigations into insect immunology, but modern techniques combined with a sound knowledge of insect physiology are now permitting rapid advances. Rather than discussing the many controversies, this review aims to point out current areas of research into cellular and 'humoral' mechanisms that might be followed up by parasitologists.

The effect of biotic and abiotic implants on the recognition of Blatta orientalis cuticular transplants by the cockroach Periplaneta americana.

The immune system of the American cockroach, Periplaneta americana, was activated to recognise as "non self" xenogeneic tissue normally treated as "self". Activation was accomplished by injecting into the insect haemocoele material known to elicit an encapsulation response such as Blaberus craniifer cuticular tissue and 6B-Sepharose beads. In these insects, cuticular skingrafts from Blatta orientalis, a closely related species of cockroach, were rejected by more than half of the recipients. There was no rejection of skingrafts by naive insects showing that the immune system of the cockroach had been triggered non-specifically to recognise tissue previously treated as "self".

Chemokinetic behaviour of insect haemocytes in vitro.

Time-lapse microphotography was used to film the locomotory behaviour of cockroach haemocytes in vitro, and the cell tracks were analysed for speed and persistence; the percentage mobilization and the diffusion rate of the population were calculated. Haemocytes are either fast locomotor or spread moving cells, or non-motile spread or rounded cells; the first three types are plasmatocytes and their behaviour is interchangeable. Approximately 20% of the cells are motile under control conditions and there is no correlation between orthokinesis and klinokinesis. If activated haemocyte lysate supernatant (HLS), a source of components of the prophenoloxidase enzyme sequence, is added to the cell monolayer, up to 80% of the cells switch to fast locomotor behaviour, rounding up and moving faster and for longer in straight lines. Neither heat-inactivated HLS nor zymosan supernatant, used to activate HLS, had any effect. If the chemokinins present in activated HLS are also released in vivo on haemocyte activation or during cuticular wounding, then they and the induced changes in haemocyte adhesion could contribute to haemocyte recruitment to sites of infection.

Hemolymph transfer as an assay for immunorecognition in the cockroach Periplaneta americana and the locust Schistocerca gregaria.

A quantitative assay for measuring the number of hemocytic nodules formed in response to foreign particles and soluble molecules has been used, in the locust Schistocerca and the cockroach Periplaneta, to investigate the response to transferred hemolymph. Xenogeneic test particles, rabbit neutrophil leukocytes, stimulate formation of nodules when injected into both insect species, compared with saline-injected controls. However, the number of nodules formed in the locust in response to cockroach hemolymph is significantly reduced compared with the response to other xenogeneic cells, and it is suggested that, in view of the strong reactivity of cockroach hemocytes to locust hemolymph and plasma, a graft-versus-host response might be occurring in the recipient locust. Whole hemolymph transferred allogeneically between Periplaneta, or xenogeneically from Blatta to Periplaneta, does not stimulate a response in the recipient. This corresponds well with results from other assays for immunorecognition of transplants and is further confirmation that allogeneic and, in some combinations xenogeneic, recognition is absent in insects.

Surface charge of insect haemocytes, examined using cell electrophoresis and cationized ferritin-binding.

Differences in the negative surface charge of haemocytes from Periplaneta americana and Schistocerca gregaria have been revealed using cell electrophoresis and cationized ferritin-binding. Although haemocyte populations from both insect species exhibit ranges of negative surface charge, both techniques show that Schistocerca haemocytes are significantly more negative than Periplaneta haemocytes. The results may help to explain why Schistocerca haemocytes adhere poorly to negative substrata, both in vitro and in vivo, and suggest that an electrostatic mechanism may be involved, at least in part, in adhesion of insect haemocytes to substrata.

Effect of substratum wettability and charge on adhesion in vitro and encapsulation in vivo by insect haemocytes.

The circulating leucocytes of insects, the haemocytes, adhere to and encapsulate foreign material that enters the insect's body cavity. The thickness of the capsule depends not only on the insect species but also on the nature of the object concerned, a fact that is of great importance to invading parasites and pathogens. In this paper, some of the factors that may stimulate haemocyte adhesion and subsequent encapsulation of the object have been investigated using abiotic materials with surfaces of different charge and wettability. The negativity and wettability of surfaces of polystyrene beads and plates can be increased by pretreatment with acid, and adhesion of haemocytes to these modified surfaces has been examined in vivo and in vitro. A similar proportion of haemocytes of the locust Schistocerca gregaria adhere to the plates in vitro, irrespective of the changes in charge and wettability, but the adhesion of haemocytes of the cockroach Periplaneta americana is proportional to the increases in both parameters. These differences in cell behaviour are reflected in vivo: cockroach haemocytes form thicker capsules around more hydrophilic and more negatively charged polystyrene beads, while locust cells encapsulate both types of surface to the same, minimal, degree. Positively and negatively charged Sepharose beads are encapsulated more thickly than are neutral beads in cockroaches; negatively charged Sepharose beads are not encapsulated at all in locusts. There are thus obvious differences between the two species in the ways in which their haemocytes respond to these modified abiotic surfaces. It is suggested that capsule thickness in vivo depends on the initial cell-substratum contact; different surfaces stimulate the cell to different extents, which in turn causes variations in the recruitment of other cells to the capsule.

Immunological recognition of cuticular transplants in insects.

Transplantation of allogeneic and xenogeneic cuticle onto the American cockroach, Periplaneta americana, was carried out in order to compare the specificity of immune recognition of these 'skin grafts' with that of implanted tissues. In order to facilitate interpretation of results, the technique of transplanting cuticle from nymphal donors onto nymphal recipients was adopted - if donor subcuticular epidermis is not recognised as 'foreign', it will grow, fuse with the recipient's epidermal sheet and will be stimulated by the recipient's hormonal signals to produce new cuticle of donor type at the next moult. Neither allogeneic cuticle nor xenogeneic cuticle from Blatta orientalis were recognised as foreign by the immune system of P. americana - dark patches of Blatta-type cuticle were produced at the graft site post-moult. Conversely, xenogeneic cuticle of Blaberus craniifer was not visible post-moult. These results corroborate those from implantation studies, that allogeneic tissues from P. americana and xenogeneic tissues from B. orientalis are immunologically compatible with P. americana, whereas xenogeneic tissue from Blaberus craniifer is incompatible. Whether this incompatibility is immunological or 'positional' has not yet been determined; the observation that xenografts from Nauphoeta cinerea do not reappear on P. americana post-moult, whereas 50% of N. cinerea implants are not recognised as 'foreign', suggests that 'positional incompatibility' (i.e. the signals responsible for formation of cuticular pattern are incorrect for the donor epidermis) may also play an important part in the rejection of N. cinerea cuticular grafts.

Humoral mechanisms in the immune response of insects to larvae of Hymenolepis diminuta (Cestoda).

Previous work has shown that larvae of Hymenolepis diminuta are not encapsulated by the haemocytes of the locust, Schistocerca gregaria, but are encapsulated by haemocytes of the cockroach, Periplaneta americana. The specificity of the cellular immune response in vivo, and of the agglutinating activity of the sera against vertebrate erythrocytes in vitro, is known to differ between the two species of insect. Accordingly, the sera of each of these species was tested for its agglutinating activity in vitro against a pure suspension of a known number of freshly-hatched oncospheres of H. diminuta; it was found that serum of P. americana strongly agglutinated oncospheres while the serum of S. gregaria had no effect. The agglutinating activity of cockroach serum was only partially removed by preadsorption with either human, rat or rabbit erythrocytes, all of which are strongly agglutinated by the serum. Although the function in vivo of this agglutinating activity directed against oncospheres is not known, it is suggested that this observation--that both the haemocytes and serum of one insect species respond to the parasite whereas the serum and haemocytes of another species apparently do not--may be of some relevance to the immune recognition mechanisms of insects.

Evasion of the insect immune response by Moniliformis dubius (Acanthocephala): further observations on the origin of the envelope.

The envelope around larvae of Moniliformis dubius appears to protect the parasite against immune recognition and encapsulation by the insect host's haemocytes. The origin of this envelope has been the subject of controversy although most evidence suggests it is parasite-derived. If host-derived, the envelope would be expected to share surface properties with host tissue. Thus, experiments were undertaken, transplanting parasites and host tissue to other insects and using haemocytic encapsulation as an assay for immune recognition, in order to compare the response to host tissue and to the parasite's envelope. Parasites without their envelopes, and pieces of tissue (ventral nerve cord) from the experimental host (the locust Schistocerca gregaria) were recognized as foreign and encapsulated in the cockroach, Periplaneta americana. The majority of parasites with their envelopes were unencapsulated or only partially encapsulated on transfer to their normal host, P. americana, indicating that the envelope does not have surface similarity to locust tissue. Cockroach-derived parasites with or without envelopes were not encapsulated in S. gregaria, suggesting that the larva itself can evade or inhibit the locust's recognition mechanism. However, since larvae which develop in S. gregaria are enclosed in an envelope, the formation of the envelope would seem to be an inherent feature of the parasite's development.

Cellular recognition of foreign-ness in two insect species, the American cockroach and the desert locust.

Encapsulation of tissue implants by haemocytes was used as the assay for immune recognition in the insects studied. If haemocytes attach to and encapsulate an object implanted within the haemocoele, it may be assumed the implant is recognized as 'not-self'; lack of encapsulation of tissue implants may be assumed to indicate lack of recognition. The American cockroach, Periplaneta americana, and the desert locust, Schistocerca gregaria, were used both as donors of tissue, and as recipients of tissue implants from various insect species. Allografts were not recognized as foreign and encapsulated by haemocytes of either S. gregaria or P. americana, a phenomenon which has been reported frequently for other insect species. Haemocytes of S. gregaria recognized and encapsulated a smaller range of xenografts compared with haemocytes of P. americana, and it appears that the acuity of immune recognition by haemocytes differs between S. gregaria and the other species tested. Reasons for the different recognition responses of P. americana and S. gregaria are suggested and discussed with particular reference to the results for Nauphoeta cinerea tissue in P. americana, and Gryllus domesticus tissue in S. gregaria, where at least half the number of implants are totally but weakly encapsulated, and it is suggested that haemocytes do not react to 'difference' unless it surpasses a certain threshold.

Evasion of the haemocytic defence reaction of certain insects by larvae of Hymenolepis diminuta (Cestoda).

Larvae of Hymenolepis diminuta develop in the haemocoele of the beetles Tribolium and Tenebrio, and of the locust Schistocerca gregaria, without being encapsulated by haemocytes. The mechanism of this evasion of the haemocytic defence reaction has been examined using various techniques. Larvae grown in culture and injected into S. gregaria have a few or no haemocytes adherent even after 8h, although latex beads injected at the same time have been thickly encapsulated. This, and results of transplanting cysticercoids and host tissue between different insect species, suggests that the surface of the larvae may bear an inherent similarity to the surface of host tissues and thus escape recognition as 'not-self' by the host's haemocytes.