Population genetics of the potentially invasive African fruit fly species, Ceratitis rosa and Ceratitis fasciventris (Diptera: Tephritidae).

A set of 10 microsatellite markers was used to survey the levels of genetic variability and to analyse the genetic aspects of the population dynamics of two potentially invasive pest fruit fly species, Ceratitis rosa and C. fasciventris, in Africa. The loci were derived from the closely related species, C. capitata. The degree of microsatellite polymorphism in C. rosa and C. fasciventris was extensive and comparable to that of C. capitata. In C. rosa, the evolution of microsatellite polymorphism in its distribution area reflects the colonization history of this species. The mainland populations are more polymorphic than the island populations. Low levels of differentiation were found within the Africa mainland area, while greater levels of differentiation affect the islands. Ceratitis fasciventris is a central-east African species. The microsatellite data over the Uganda/Kenya spatial scale suggest a recent expansion and possibly continuing gene flow within this area. The microsatellite variability data from C. rosa and C. fasciventris, together with those of C. capitata, support the hypothesis of an east African origin of the Ceratitis spp.

Comparative analysis of microsatellite loci in four fruit fly species of the genus Ceratitis (Diptera: Tephritidae).

The possibility to cross-species amplify microsatellites in fruit flies of the genus Ceratitis was tested with the polymerase chain reaction (PCR) by analysing 23 Ceratitis capitata (Wiedemann) microsatellite markers on the genomic DNA of three other economically important, congeneric species: C. rosa (Karsch), C. fasciventris (Bezzi) and C. cosyra (Walker). Twenty-two primer pairs produced amplification products in at least one of the three species tested. The majority of the products were similar, if not identical in size to those expected in C. capitata. The structures of the repeat motifs and their flanking sequences were examined for a total of 79 alleles from the three species. Sequence analysis revealed the same repeat type as the homologous C. capitata microsatellites in the majority of the loci, suggesting their utility for population analysis across the species range. A total of seven loci were differentially present/absent in C. capitata, C. rosa, C. fasciventris and C. cosyra, suggesting that it may be possible to differentiate these four species using a simple sequence repeat-based PCR assay. It is proposed that medfly-based microsatellite markers could be utilized in the identification and tracing of the geographical origins of colonist pest populations of the four tested species and in the assessment of their risk and invasive potentials; thereby assisting regulatory authorities in implementing quarantine restrictions and other pest control measures.

Characterization of protective antigens from the midgut of Amblyomma variegatum ticks.

Separation of midgut membrane proteins from the tick, Ambylomma variegatum, using a nonionic detergent (Triton X-114), resulted in two protein fractions, namely DET (detergent) and AQ (aqueous). In immunoblotting analysis with polyclonal antibodies against these fractions, 4 proteins (Mr approximately 27,000, 67,000, 86,000 and 95,000,) and 2 proteins (M, approximately 54,000 and 67,000) were detected in the DET and AQ fractions, respectively. Three of the DET fraction proteins Mr approximately 27,000, 67,000 and 95,000 were glycosylated since they bound to the lectin, concanavalin A. In 2-dimensional gel electrophoresis, the AQ and DET fraction proteins were found to be acidic in nature. In a series of bioassay experiments, rabbits were first immunised with both DET and AQ fractions and then infested with ticks. The egg batch weights of these ticks were reduced by 50% compared to control ticks. Furthermore, there was a significant reduction in the hatchability of eggs laid by ticks fed on rabbits previously immunised with both DET (14%) and AQ (33%) fractions. Based on the egg hatchability, the reproductive capacity of ticks was reduced by 77 and 48% by DET and AQ fractions, respectively.

The feeding behaviour and Plasmodium infection of Anopheles mosquitoes in southern Ethiopia in relation to use of insecticide-treated livestock for malaria control.

Anopheles arabiensis and An. quadriannulatus species B mosquitoes were collected at sites of human and livestock housing and analysed for blood feeding patterns and infection with malaria sporozoites. A low percentage of human blood meals at some sites suggested that zooprophylaxis could be effective in reducing challenge from Plasmodium falciparum.

Cloning and expression of a Bacillus thuringiensis (L1-2) gene encoding a crystal protein active against Glossina morsitans morsitans and Chilo partellus.

A local isolate of Bacillus thuringiensis,designated L1-2, that is toxic to Chilo partellus was found to be toxic to the adult tsetse fly, Glossina morsitans morsitans. The delta-endotoxin crystals derived from the isolate gave a major protein band with a molecular weight of Mr 130,000-140,000 on denaturing polyacrylamide gel electrophoresis. The sequence of the cloned gene was found to be similar to that of the B. thuringiensis subsp. kurstaki HD-73 cryIA(c) gene, having one amino acid difference at position 148 and four additional DNA differences.

Effects of juvenile hormone treatment on phase changes and pheromone production in the desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae).

The roles of juvenile hormone III (JH III) on phase changes and pheromone production were examined in laboratory-reared gregarious desert locust, Schistocerca gregaria (Forskal). The hormone was applied to 5th instar nymphs and newly emerged adult locusts. Generally, the 5th instar nymphs exhibited a higher sensitivity to hormone treatments than the adults. Hormone applications inhibited pheromone production (as measured by the amounts of phenylacetonitrile released). In addition, JH III had a significant effect on the external colouration and absorbance ratios of the haemolymph pigments. It is concluded that the effects of exogenous JH III on gregarious locusts represent a shift towards the solitarious phase.

The effect of host blood in the in vitro transformation of bloodstream trypanosomes by tsetse midgut homogenates.

Midgut homogenates prepared from Glossina morsitans morsitans, that had previously been fed on different host blood samples, were tested for their abilities to transform bloodstream Trypanosoma brucei into procyclic (midgut) forms in vitro. Compared to rat and goat blood samples, eland blood had the least capacity to support trypanosome transformation, whereas buffalo blood showed intermediate capacity. Fractionation of rat blood showed the importance of the cellular portion since both rat and eland red blood cells (RBCs) supported the process. Virtually no transformation was observed in rat and eland plasma or serum fractions. Suspending rat blood cells in eland plasma led to a reduction in parasite transformation rates. Further experiments showed that the RBC membranes were also capable of supporting the process. These results clearly show the important role played by blood, especially the red blood cells, in the transformation of bloodstream trypanosomes. In addition, the low transformation rates observed in eland blood is due to an inhibitory factor(s) present in the plasma fraction.

Properties of a blood-meal-induced midgut lectin from the tsetse fly Glossina morsitans.

The properties of a blood-meal-induced lectin (agglutinin) from the midgut of Glossina morsitans capable of agglutinating Trypanosoma brucei were studied in vitro. The midgut homogenate from flies that had been fed twice had the highest agglutination activity, followed by that from the once-fed flies and that from the unfed insects. As compared with the bloodstream-form trypanosomes, a much lower concentration of the midgut homogenate was required for agglutination of the procyclic parasites. Furthermore, the agglutination process was specifically inhibited by D-glucosamine. Soybean trypsin inhibitor abrogated agglutination of the bloodstream-form parasites, whereas the procyclics were unaffected. The agglutination process was temperature-sensitive, with little activity being evident between 4 degrees and 15 degrees C. Similarly, heating the midguts to 60 degrees-100 degrees C led to loss of activity. When the midgut homogenate was separated by anion-exchange chromatography, the agglutination activity co-eluted with trypsin activity at approximately 50% NaCl. These results suggest a very close relationship between midgut trypsin-like enzyme and the agglutinin. Since successful agglutination of bloodstream-form trypanosomes requires protease activity, it may be that the enzyme cleaves off some surface molecules on the parasite surface, thus exposing the lectin-binding sites.

Purification and characterization of a midgut lectin-trypsin complex from the tsetse fly Glossina longipennis.

A blood-meal-induced lectin (agglutinin) with proteolytic activity was isolated from midgut extracts of Glossina longipennis by a two-step procedure involving anion-exchange chromatography. It is a glycoprotein [native molecular weight (M(r) 61,000 +/- 3000 da) composed of two noncovalently-linked subunits designated alpha (M(r), approximately 27,000 da) and beta (M(r), approximately 33,000 da). The trypsin activity and the glycosyl residues were present on the alpha- and beta-subunits, respectively. The native protein was capable of agglutinating both bloodstream-form and procyclic trypanosomes as well as rabbit red blood cells. This activity was strongly inhibited by D-glucosamine and weakly inhibited by N-acetyl-D-glucosamine. Similarly, soybean trypsin inhibitor abrogated agglutination of bloodstream-form parasites, whereas the procyclics were unaffected. The agglutination activity was sensitive to temperatures above 40 degrees C but was unaffected by chelators of metal ions. Antibodies raised against the protein were used in immunoblotting experiments to show the presence of a similar protein in several members of the Glossina species. However, no cross-reactivity was detected with midgut extracts prepared from sandflies, mosquitoes, or stable flies. It is proposed that this molecule might play an important role in differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms.

Temporal synthesis of cuticle proteins during larval development in Glossina morsitans.

1. Larval development in Glossina species occurs in utero with the mature third instar larva being deposited after a developmental period of 7 days. 2. In this study, the patterns of cuticular protein synthesis during larval development were analysed by two-dimensional gel electrophoresis. 3. From the results, four types of cuticle proteins were identified: those specific to larval, pupal and adult cuticles, and others common to all the stages. 4. Few cuticular proteins were synthesized between the first and second larval instars. By the third larval instar (two days before larviposition), a large number of proteins (Mr < or = 30 kDa) were induced. These proteins persisted up to the brown pupal stage and showed a rapid decline thereafter. Most of the proteins with molecular weights Mr < or = 30 kDa were undetectable at apolysis (5 days after larviposition). 5. By day 15 of the pupal stage, the number of cuticle proteins was very small. The protein profile during the pupal stages remained relatively constant. This was probably due to the fact that the pupal cuticle does not provide any protection since it is itself enclosed at all times within the protective puparium.

Inhibition of Glossina morsitans midgut trypsin activity by D-glucosamine.

The effect of the amino sugar D-glucosamine on trypsin in crude midgut homogenates of Glossina morsitans morsitans was studied in vitro. The results showed that the midgut trypsin was specifically and competitively inhibited by D-Glucosamine. Glucose, fructose, mannose, inositol, galactose, galactosamine, N-acetyl-D-glucosamine, and methyl-alpha-D-glucosamine were ineffective as inhibitors, even at concentrations exceeding 600 mM. D-glucosamine also had a similar inhibitory effect on bovine pancreatic trypsin. In both cases, the inhibition was incomplete as shown by nonlinear Dixon plots. The Michaelis and inhibition constants estimated for the midgut trypsin were 41 +/- 2 microM and 68 +/- 3 mM, respectively. These results suggest that the susceptibility of tsetse flies to trypanosome infection, which is associated with high midgut glucosamine levels, could be due to inhibition of trypsin or trypsin-like enzymes by this sugar.

Studies on tsetse midgut factors that induce differentiation of blood-stream Trypanosoma brucei brucei in vitro.

An in vitro system for studying the transformation of bloodstream forms of Trypanosoma brucei brucei into procylic (midgut) forms is described. In this system, transformation of the parasites was stimulated by Glossina morsitans morsitans midgut homogenates at 27 degrees C but not at 4 degrees C. The transformation-stimulating capacity was irreversibly destroyed by heating the midgut homogenates at 60 degrees C for 1 h. A correlation was established between the transformation activity of the midgut homogenates and trypsin activity. The protease inhibitors (soybean trypsin inhibitor and N-p-tosyl-L-lysine-chloromethyl-ketone) inhibited trypsin activity and completely blocked the transformation of the parasites. Furthermore, the midgut homogenates could induce transformation only in the presence of blood. These results provide evidence for the involvement of trypsin or trypsin-like enzymes within the tsetse midgut in stimulation of the transformation of bloodstream trypanosomes.

Isolation and properties of 600-kDa and 23-kDa haemolymph proteins from the tsetse fly, Glossina morsitans: their possible role as biological insecticides.

The haemolymph of the tsetse fly, Glossina morsitans morsitans, contains a high (lipophorin) and a low molecular weight protein of high densities, 1.11 and 1.29 g/ml, respectively. The purification of the proteins was achieved by a combination of density gradient ultracentrifugation and reported gel permeation chromatography. The lipophorin is of high molecular weight (M(r) integral of 600,000) and consists of two apoproteins, apolipophorin I (M(r) integral of 250,000) and apolipophorin II (M(r) integral of 80,000) both of which are glycosylated. Lipophorin also has a pI of 6.1. However, electrophoresis under non-denaturing and denaturing conditions showed the low molecular weight protein to be a single polypeptide chain (M(r) integral of 23,000). Amino acid analysis revealed a relatively high content of the acidic amino acids as well as serine and glycine. The protein contained lipids as shown by Sudan Black staining but was unglycosylated. Using rabbit antiserum against the isolated protein in immunodiffusion and immunoblotting experiments, no cross-reactivity was detected with haemolymph samples from insects representing six orders. In conclusion, the finding of lipophorin suggests that, although flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements. However, the results for the low molecular weight protein indicate that the protein is unique to Glossina, suggesting that it may have an important role in the physiology of this insect and is therefore a significant target for vector management.

Inhibitory effect of Trypanosoma brucei brucei on Glossina morsitans midgut trypsin in vitro.

The ability of Trypanosoma brucei brucei to inhibit trypsin or trypsin-like enzymes in crude midgut homogenates of Glossina morsitans morsitans was studied in vitro. The isolated parasites caused a concentration-dependent decrease in midgut trypsin activity. Furthermore, trypanosomes lysed by repeated freeze-thawing had a similar effect on trypsin activity. In both cases, the inhibition by either intact or lysed parasites was partial as revealed by Dixon plots. Similarly, trypanosome membrane proteins stoichiometrically inhibited trypsin activity, suggesting that the enzyme interacts specifically with a moiety on the parasite surface. The Km and Ki values obtained in this case were 35 microM and 0.18 mg/ml, respectively. These results suggest that one of the ways in which trypanosomes overcome the hostile tsetse-fly midgut barrier involves the inhibition of enzyme activity.

Lipophorin from the tsetse fly, Glossina morsitans morsitans.

1. Lipophorin was isolated from the haemolymph of adult tsetse fly, Glossina morsitans morsitans, by ultracentrifugation in a potassium bromide density gradient. 2. The tsetse fly lipophorin (Mr congruent to 600,000) has a density of congruent to 1.11 g/ml and consists of two apoproteins, apolipophorin-I (apoLp-I, Mr congruent to 250,000) and apolipophorin-II (apoLp-II, Mr congruent to 80,000), both of which are glycosylated as shown by staining with periodate-Schiff reagent. The protein complex is composed of 49% protein and 51% lipids. 3. The finding of lipophorin in tsetse fly haemolymph suggests that, although these flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements.

Structural studies on the major milk gland protein of the tsetse fly, Glossina morsitans morsitans.

1. The major protein in the milk gland secretions of the tsetse fly, Glossina morsitans morsitans, was isolated by a combination of gel permeation chromatography and crystallization. 2. It has a native Mr approximately 47,000 and is composed of two identical polypeptide chains (Mr approximately 21,000) as determined by chemical cross-linking studies. The protein has no covalently-bound carbohydrates or lipids. Amino acid analysis of the protein revealed relatively high amounts of the aromatic amino acids, tyrosine (9.1 mol.%) and phenylalanine (8.5 mol.%). Immunoblotting experiments using antiserum against the protein revealed no cross-reactivity with any other milk proteins. 3. Quantitation of the protein during the pregnancy cycle showed that synthesis of the protein by the milk glands of adult female flies starts as the larva moults into second instar and rapidly declines as it matures into third instar. 4. It is proposed that the major milk gland protein could provide essential amino acids needed for the puparium formation.

Juvenile hormone receptors in insect larval epidermis: identification by photoaffinity labeling.

Tritiated photoaffinity analogs of the natural lepidopteran juvenile hormones, JH I and II [epoxy[3H]bishomofarnesyl diazoacetate ([3H]EBDA) and epoxy[3H]homofarnesyl diazoacetate ([3H]EHDA)], and of the JH analog methoprene [[3H]methoprene diazoketone ([3H]MDK)] were synthesized and used to identify specific JH binding proteins in the larval epidermis of the tobacco hornworm (Manduca sexta). EBDA and EHDA specifically photolabeled a 29-kDa nuclear protein (pI 5.8). This protein and a second 29-kDa protein (pI 6.0) were labeled by MDK, but excess unlabeled methoprene or MDK only prevented binding to the latter. These 29-kDa proteins are also present in larval fat body but not in epidermis from either wandering stage or allatectomized larvae, which lack high-affinity JH binding sites. A 29-kDa nuclear protein with the same developmental specificity as this JH binder bound the DNA of two larval endocuticle genes. A 38-kDa cytosolic protein was also specifically photolabeled by these photoaffinity analogs. The 29-kDa nuclear protein is likely the high-affinity receptor for JH that mediates its genomic action, whereas the 38-kDa cytosolic protein may serve as an intracellular carrier for these highly lipophilic hormones and hormone analogs.

Nuclear binding sites for juvenile hormone and its analogs in the epidermis of the tobacco hornworm.

Juvenile hormones (JH) are sesquiterpene derivatives that regulate both morphogenetic and reproductive development in insects. The larval epidermis of the tobacco hornworm, Manduca sexta, was found to take up both 3H-JH I and a biologically active JH analog, [125I]iodovinylmethoprenol (IVMA), from the incubation medium with 33% of the label going to the nucleus in both cases. An exchange assay using isolated nuclei showed the presence of two binding sites with approximate KD values of 7 and 88 nM for JH I and 4 and 59 nM for IVMA. There were about 10,000 of the high affinity sites per nucleus. The binding of both hormones was sensitive to pH and Pronase digestion. In competition studies, JH II and JH III competed for 3H-JH I binding sites, whereas IVMA, hydroprene, and methoprene did not. In similar studies, methoprene and hydroprene competed for [125I]IVMA binding sites but JH I, JH II, and JH III were all ineffective. These results are consistent with the presence of specific and distinct binding sites for JH and IVMA in these nuclei.

Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.

The egg of Manduca sexta contains a very high density lipophorin (VHDLp-E; Mr approximately equal to 4.14 x 10(5), rho = 1.238 g/ml) that is derived from the high density lipophorin (HDLp-A; Mr approximately equal to 7.63 x 10(5), rho = 1.076 g/ml) of the hemolymph. The selective uptake of HDLp-A into the egg and its subsequent conversion to VHDLp-E was studied both in vivo and in vitro. Upon entering the egg, an estimated 530 mol of lipid were stripped from each mol of HDLp-A, and 68% of the diacylglycerol fraction was converted to triacylglycerol. In addition, the two molecules of the low molecular weight apolipoprotein, apolipophorin-III, of HDLp-A were dissociated from the lipophorin particle. The VHDLp-E thus formed consisted of 80% protein and 20% lipid, 75% of which was phospholipid. HDLp-A labeled in vivo with [35S]methionine in its apoprotein moiety was injected into females at the onset of egg development, and its incorporation in a series of follicles at different stages of growth was measured. There was increased accumulation of [35S]HDLp-A in the follicles as they matured. The apoproteins of [35S]HDLp-A were not hydrolyzed when the particle was internalized by the follicle. In the accompanying paper we have presented the evidence that the apoproteins of HDLp-A are retained in the follicles (Kawooya, J.K., and Law, J.H. (1988) J. Biol. Chem. 263, 8748-8753).