Peter F. Billingsley

Partial characterization of oligosaccharides expressed on midgut microvillar glycoproteins of the mosquito, Anopheles stephensi Liston.

Midguts of the malaria-transmitting mosquito, Anopheles stephensi, were homogenized and microvillar membranes prepared by calcium precipitation and differential centrifugation. Oligosaccharides present on the microvillar glycoproteins were identified by lectin blotting before and after in vitro and in situ treatments with endo- and exo-glycosidases. Twenty-eight glycoproteins expressed a structurally restricted range of terminal sugars and oligosaccharide linkages. Twenty-three glycoproteins expressed oligomannose and/or hybrid N-linked oligosaccharides, some with alpha1-6 linked fucose as a core residue. Complex-type N-linked oligosaccharides on eight glycoproteins all possessed terminal N-acetylglucosamine, and alpha- and beta-linked N-acetylgalactosamine. Eight glycoproteins expressed O-linked oligosaccharides all containing N-acetylgalactosamine with or without further substitutions of fucose and/or galactose. Galactosebeta1-3/4/6N-acetylglucosamine-, sialic acidalpha2-3/6galactose-, fucosealpha1-2galactose- and galactosealpha1-3galactose- were not detected. Terminal alpha-linked N-acetylgalactosamine residues on N-linked oligosaccharides are described for the first time in insects. The nature and function of these midgut glycoproteins have yet to be identified, but the oligosaccharide side chains are candidate receptors for ookinete binding and candidate targets for transmission blocking strategies.

Vector-parasite interactions for vaccine development

The ingestion of blood by arthropod vectors of disease can be exploited in order to either kill the vector or render it incapable of disease transmission. This paper examines some approaches to identifying target molecules of vector origin, against which immunisation could result in blocking parasite transmission. Manipulation of the blood meal of vectors through such techniques as membrane feeding can help identify true target sites for attack, but just as useful, can identify structures or molecules that play no significant role in parasite development. Examples, mostly derived from the interactions between the malaria parasite, Plasmodium, and the mosquito midgut, illustrate the real need to understand the multiple aspects of vector-parasite interactions before they can be exploited for control purposes. The approaches outlined are however applicable directly to any vector-borne disease. Careful examination of the parasite life cycle in the vector, and comparisons with other parasites, vectors, non-vector insects and analogous vertebrate systems (the latter being often relatively well advanced) can result in the identification of specific and definable interactions which can then be further developed for vaccine purposes.

Kinetics of Expression of Two Major Plasmodium berghei Antigens in the Mosquito Vector, Anopheles stephensi

ABSTRACT. Expression of a 21 kDa determinant (Pbs21), first detected on the surface of ookinetes, and of the circumsporozoite protein (CSP) was studied by immunofluorescence and Western blots during the developmental cycle of Plasmodium berghei in the mosquito A nopheles stephensi. The expression of Pbs21 was predominantly localised on the ookinete surface one day after the infectious blood meal, and thereafter reactivity declined to a minimum on days 2 and 3, the time of onset of oocyst development. A gradual increase in fluorescence was observed on the oocysts from day 6 that was retained until day 17 post‐infection. In contrast, sporozoites released from oocysts or salivary glands showed little or no antibody labelling with anti‐Pbs21. Circumsporozoite protein was not detectable in any rnidgut preparations until 5–6 days after feeding, when reactivity was observed against immature oocysts. Expression then continued and increased throughout oocyst and sporozoite development. Western blots confirmed that Pbs21 was expressed minimally during the oocyst development but was not detectable in sporozoites. Co‐localisation of anti‐Pbs21 and anti‐CSP monoclonal antibodies to the 50 kDa and 60 kDa bands in Western blots of sporozoite suggests immunological cross‐reactivity between the CSP and the anti‐21 kDa antibodies.

Detection of mature malaria infections in live mosquitoes

A method has been developed which detects malaria parasites in the salivary glands of live Anopheles stephensi. The method exploits the sugar feeding behaviour of the mosquito and requires only routine Western blotting techniques on nitrocellulose membrane (NCM). Infectivity can be determined without any direct manipulation of individual mosquitoes. Female A. stephensi were infected with the rodent malaria parasite, Plasmodium berghei, and after 14-16 d were starved of fructose overnight (12-18 h), then resupplied with fructose presented through a small piece of NCM. Mosquitoes were allowed to probe the membrane for several hours; the NCM was then removed and subjected to a standard immunoblotting protocol using an anti-P. berghei circumsporozoite protein (CSP) monoclonal antibody as the primary reagent, and a horseradish peroxidase-coupled secondary antibody. NCMs taken from cages containing infected mosquitoes showed a variable number of small black dots where individual females had probed and deposited either CSP or sporozoites. Infectivity could be detected easily from 13-14 d after feeding, and in as few as 10 mosquitoes at 19 d after infection; in one instance, infection in a single mosquito was clearly determined. After blocking with goat serum, the NCMs could be stored for 3-4 months and still provided positive reactions, offering some potential for applicability to field research studies.

Approaches to vector control: new and trusted: 2. Molecular targets in the insect midgut

The midgut of blood-feeding insects is an important site for the activity of antibodies and drugs ingested with the blood meal. These agents can be directed at molecular targets in the midgut, and may affect the insect directly by reducing its fitness, or indirectly by blocking transmission of a disease organism to the vector. Both of these result in eventual disease control. Immunization with crude vector or parasite preparations can result in isolation of vaccine candidates which are very effective but often of unknown function. Conversely, by examining carefully the vectors biology and its interactions with the parasite, it is possible to identify various physiological or cellular systems and vector-parasite interactions that can be interfered with. Examples of both approaches are presented in this paper. Anti-vector vaccines offer tremendous potential for disease control, as they can affect the parasite reproductive rate in a number of ways--reducing vector longevity, fecundity or competence. This is most striking if life expectancy of the vector is reduced to a period less than the extrinsic life cycle of the parasite. The rationale often presented for examining molecular systems in vectors is their control potential; it is clearly appropriate to re-examine many of these systems and realistically assess their applicability.The midgut of blood-feeding insects is an important site for the activity of antibodies and drugs ingested with the blood meal. These agents can be directed at molecular targets in the midgut, and may affect the insect directly by reducing its fitness, or indirectly by blocking transmission of a disease organism to the vector. Both of these result in eventual disease control. Immunization with crude vector or parasite preparations can result in isolation of vaccine candidates which are very effective but often of unknown function. Conversely, by examining carefully the vectors biology and its interaction with the parasite, it is possible to identify various physiological or cellular systems and vector-parasite interactions that can be interfered with. Examples of both approaches are presented in this paper. Anti-vector vaccines offer tremendous potential for disease control, as they can affect the parasite reproductive rate in a number of ways—reducing vector longevity, fecundity or competence. This is most striking if life expectancy of the vector is reduced to a period less than the extrinsic life cycle of the parasite. The rationale often presented for examining molecular systems in vectors is their control potential; it is clearly appropriate to re-examine many of these systems and realistically assess their applicability.

Ouabain-sensitive Na+/K+-ATPase activity in the reservoir zone of the midgut of Stomoxys calcitrans (diptera: muscidae)

Abstract Na + /K + -ATPase activity was studied in the midgut of Stomoxys calcitrans . Both male and female flies possessed a ouabain-sensitive Na + /K + -ATPase in the reservoir zone of the midgut, while the digestive regions showed no ouabain-sensitive activity. In the reservoir zone, > 90% of the total ATPase activity was sensitive to inhibition by ouabain with an IC 50 of 8.8 × 10 −7 ± 1.6 × 10 −7 M and maximal inhibition occurring at 10 −5 M. This ouabain-sensitive Na + /K + -ATPase was activated maximally at a Mg 2+ : ATP ratio of 1:1, with a K m of 0.31 mM and a V max of 14.0 μmol Pi mg protein −1 min −1 for ATP. Maximal activation was reached at 12 mM K + ( K m = 0.18 mM), while activation with Na + showed an increase up to 75 mM ( K m = 22.65 mM). The optimal K + :Na + ratio was 1:77. The ouabain-sensitive enzyme was inhibited by Ca 2+ with an IC 50 of 0.1 ± 0.01 mM and was optimally active at pH 7.2. The minor ouabain-insensitive fraction was unaffected by Na + K, + Mg, 2+ or Ca 2+ but showed some dependence on ATP. The kinetic and ouabain-inhibition characteristics of the stablefly ouabain-sensitive Na + /K + -ATPase appear to correspond most closely with those of the mammalian α3 isoform. The demonstration of a ouabain-sensitive Na + /K + -ATPase being a major ATPase in the stablefly reservoir zone is consistent with the hypothesis that this region is actively involved in post-feeding ion transport and water regulation.

Mosquito cell line glycoproteins: an unsuitable model system for the Plasmodium ookinete-mosquito midgut interaction?

BackgroundMosquito midgut glycoproteins may act as key recognition sites for the invading malarial ookinete. Effective transmission blocking strategies require the identification of novel target molecules. We have partially characterised the surface glycoproteins of two cell lines from two mosquito species; Anopheles stephensi and Anopheles gambiae, and investigated the binding of Plasmodium berghei ookinetes to carbohydrate ligands on the cells. Cell line extracts were run on SDS-PAGE gels and carbohydrate moieties determined by blotting against a range of biotinylated lectins. In addition, specific glycosidases were used to cleave the oligosaccharides.ResultsAn. stephensi 43 and An. gambiae 55 cell line glycoproteins expressed oligosaccharides containing oligomannose and hybrid oligosaccharides, with and without α1-6 core fucosylation; N-linked oligosaccharides with terminal Galβ1-3GalNAc or GalNAcβ1-3Gal; O-linked α/βGalNAc. An. stephensi 43 cell line glycoproteins also expressed N-linked Galβ1-4R and O-linked Galβ1-3GalNAc. Although P. berghei ookinetes bound to both mosquito cell lines, binding could not be inhibited by GlcNAc, GalNAc or Galactose.ConclusionsAnopheline cell lines displayed a limited range of oligosaccharides. Differences between the glycosylation patterns of the cell lines and mosquito midgut epithelial cells could be a factor why ookinetes did not bind in a carbohydrate inhibitable manner. Anopheline cell lines are not suitable as a potential model system for carbohydrate-mediated adhesion of Plasmodium ookinetes.

2. Molecular targets in the insect midgut

The midgut of blood-feeding insects is an important site for the activity of antibodies and drugs ingested with the blood meal. These agents can be directed at molecular targets in the midgut, and may affect the insect directly by reducing its fitness, or indirectly by blocking transmission of a disease organism to the vector. Both of these result in eventual disease control. Immunization with crude vector or parasite preparations can result in isolation of vaccine candidates which are very effective but often of unknown function. Conversely, by examining carefully the vectors biology and its interactions with the parasite, it is possible to identify various physiological or cellular systems and vector-parasite interactions that can be interfered with. Examples of both approaches are presented in this paper. Anti-vector vaccines offer tremendous potential for disease control, as they can affect the parasite reproductive rate in a number of ways--reducing vector longevity, fecundity or competence. This is most striking if life expectancy of the vector is reduced to a period less than the extrinsic life cycle of the parasite. The rationale often presented for examining molecular systems in vectors is their control potential; it is clearly appropriate to re-examine many of these systems and realistically assess their applicability.The midgut of blood-feeding insects is an important site for the activity of antibodies and drugs ingested with the blood meal. These agents can be directed at molecular targets in the midgut, and may affect the insect directly by reducing its fitness, or indirectly by blocking transmission of a disease organism to the vector. Both of these result in eventual disease control. Immunization with crude vector or parasite preparations can result in isolation of vaccine candidates which are very effective but often of unknown function. Conversely, by examining carefully the vectors biology and its interaction with the parasite, it is possible to identify various physiological or cellular systems and vector-parasite interactions that can be interfered with. Examples of both approaches are presented in this paper. Anti-vector vaccines offer tremendous potential for disease control, as they can affect the parasite reproductive rate in a number of ways—reducing vector longevity, fecundity or competence. This is most striking if life expectancy of the vector is reduced to a period less than the extrinsic life cycle of the parasite. The rationale often presented for examining molecular systems in vectors is their control potential; it is clearly appropriate to re-examine many of these systems and realistically assess their applicability.

Society meeting Meeting at Manson House, London, 3 March 1993 jointly with the Royal Entomological Society2. Molecular targets in the insect midgut

The midgut of blood-feeding insects is an important site for the activity of antibodies and drugs ingested with the blood meal. These agents can be directed at molecular targets in the midgut, and may affect the insect directly by reducing its fitness, or indirectly by blocking transmission of a disease organism to the vector. Both of these result in eventual disease control. Immunization with crude vector or parasite preparations can result in isolation of vaccine candidates which are very effective but often of unknown function. Conversely, by examining carefully the vectors biology and its interactions with the parasite, it is possible to identify various physiological or cellular systems and vector-parasite interactions that can be interfered with. Examples of both approaches are presented in this paper. Anti-vector vaccines offer tremendous potential for disease control, as they can affect the parasite reproductive rate in a number of ways--reducing vector longevity, fecundity or competence. This is most striking if life expectancy of the vector is reduced to a period less than the extrinsic life cycle of the parasite. The rationale often presented for examining molecular systems in vectors is their control potential; it is clearly appropriate to re-examine many of these systems and realistically assess their applicability.The midgut of blood-feeding insects is an important site for the activity of antibodies and drugs ingested with the blood meal. These agents can be directed at molecular targets in the midgut, and may affect the insect directly by reducing its fitness, or indirectly by blocking transmission of a disease organism to the vector. Both of these result in eventual disease control. Immunization with crude vector or parasite preparations can result in isolation of vaccine candidates which are very effective but often of unknown function. Conversely, by examining carefully the vectors biology and its interaction with the parasite, it is possible to identify various physiological or cellular systems and vector-parasite interactions that can be interfered with. Examples of both approaches are presented in this paper. Anti-vector vaccines offer tremendous potential for disease control, as they can affect the parasite reproductive rate in a number of ways—reducing vector longevity, fecundity or competence. This is most striking if life expectancy of the vector is reduced to a period less than the extrinsic life cycle of the parasite. The rationale often presented for examining molecular systems in vectors is their control potential; it is clearly appropriate to re-examine many of these systems and realistically assess their applicability.