Responses of tsetse flies, Glossina morsitans morsitans and Glossina pallidipes, to baits of various size.

Recent studies of Palpalis group tsetse [Glossina fuscipes fuscipes (Diptera: Glossinidae) in Kenya] suggest that small (0.25 × 0.25 m) insecticide-treated targets will be more cost-effective than the larger (≥1.0 × 1.0 m) designs currently used to control tsetse. Studies were undertaken in Zimbabwe to assess whether small targets are also more cost-effective for the Morsitans group tsetse, Glossina morsitans morsitans and Glossina pallidipes. Numbers of tsetse contacting targets of 0.25 × 0.25 m or 1.0 × 1.0 m, respectively, were estimated using arrangements of electrocuting grids which killed or stunned tsetse as they contacted the target. Catches of G. pallidipes and G. m. morsitans at small (0.25 × 0.25 m) targets were, respectively, ∼1% and ∼6% of catches at large (1.0 × 1.0 m) targets. Hence, the tsetse killed per unit area of target was greater for the larger than the smaller target, suggesting that small targets are not cost-effective for use against Morsitans group species. The results suggest that there is a fundamental difference in the host-orientated behaviour of Morsitans and Palpalis group tsetse and that the former are more responsive to host odours, whereas the latter seem highly responsive to visual stimuli.

Oviposition responses of Anopheles gambiae s.s. (Diptera: Culicidae) and identification of volatiles from bacteria-containing solutions.

In this study, a dual-choice oviposition bioassay was used to screen responses of gravid An. gambiae toward 17 bacterial species, previously isolated from Anopheles gambiae s.l. (Diptera: Culicidae) midguts or oviposition sites. The 10 isolates from oviposition sites have been identified by phylogenetic analyses of their 16S rRNA genes. Eight of the 10 isolates were gram-positive, out of which six belonged to the Bacilli class. Solid phase microextraction and gas chromatography coupled to mass spectrometry (GC-MS) were used to identify the volatiles emitted from the bacterial isolates. Aromatic and aliphatic alcohols, aliphatic ketones, alkylpyrazines, dimethyl oligosulfides, and indole were among the chemical compounds identified from the headspace above bacteria-containing saline. The mosquitoes laid significantly more eggs in six of the bacteria-containing solutions compared with the sterile solution. These six bacteria did not emit any compounds in common that could explain the positive oviposition response. Instead, the bacteria were grouped according to principal component analysis (PCA) based on the relative amounts of volatiles emitted. The PCA-plots facilitated the identification of 13 putative oviposition attractants for An. gambiae mosquitoes.

Transstadial and horizontal transfer of bacteria within a colony of Anopheles gambiae (Diptera: Culicidae) and oviposition response to bacteria-containing water.

In a paratransgenic approach, genetically modified bacteria are utilized to kill the parasite in the vector gut. A critical component for paratransgenics against malaria is how transgenic bacteria can be introduced and then kept in a mosquito population. Here, we investigated transstadial and horizontal transfer of bacteria within an Anopheles gambiae mosquito colony with the focus on spiked breeding sites as a possible means of introducing bacteria to mosquitoes. A Pantoea stewartii strain, previously isolated from An. gambiae, marked with a green fluorescent protein (GFP), was introduced to mosquitoes in different life stages. The following life stages or older mosquitoes in the case of adults were screened for bacteria in their guts. In addition to P. stewartii other bacteria were isolated from the guts: these were identified by 16S rRNA sequence analysis and temporal temperature gradient gel electrophoresis (TTGE). Bacteria were transferred from larvae to pupae but not from pupae to adults. The mosquitoes were able to take up bacteria from the water they emerged from and transfer the same bacteria to the water they laid eggs in. Elizabethkingia meningoseptica was more often isolated from adult mosquitoes than P. stewartii. A bioassay was used to examine An. gambiae oviposition responses towards bacteria-containing solutions. The volatiles emitted from the solutions were sampled by headspace-solid phase microextraction (SPME) and identified by gas chromatography and mass spectrometry (GC-MS) analysis. P. stewartii but not E. meningoseptica mediated a positive oviposition response. The volatiles emitted by P. stewartii include indole and 3-methyl-1-butanol, which previously have been shown to affect An. gambiae mosquito behaviour. E. meningoseptica emitted indole but not 3-methyl-1-butanol, when suspended in saline. Taken together, this indicates that it may be possible to create attractive breeding sites for distribution of genetically modified bacteria in the field in a paratransgenic approach against malaria. Further research is needed to determine if the bacteria are also transferred in the same way in nature.

Re-introducing bacteria in mosquitoes--a method for determination of mosquito feeding preferences based on coloured sugar solutions.

In this study, sugar-feeding was investigated as a possible means of re-introducing bacteria into mosquito midguts with the aim of identifying bacteria that are suitable for creating paratransgenic mosquitoes. In a paratransgenic approach, bacteria are utilised to deliver effector molecules capable of inhibiting pathogen development in the midgut of the vector. To determine if mosquitoes discriminate between sterile sugar solutions and sugar solutions with bacteria, a method for screening mosquito feeding preferences was developed. This method was tested for Aedes aegypti, Anopheles arabiensis and An. gambiae s.s. mosquitoes and is based on a dual-choice test of solutions labelled with food dyes. Three different tests (dye/colour detection, sugar detection and sugar-concentration detection) were performed to evaluate the method, after which bacteria previously isolated from mosquitoes were used in the experiments. It was shown that mosquitoes do not discriminate between sugar solutions with or without these bacteria indicating that sugar-feeding is a possible means to introduce bacteria into mosquitoes. Furthermore, two different setups of the method were used, enabling us to differentiate between tactile/taste and olfactory responses. The method described in this paper is easy to use, cost-effective and allows broad screening of mosquito sugar-feeding preferences.

Adjuvant activities of immune response modifier R-848: comparison with CpG ODN.

R-848 and imiquimod belong to a class of immune response modifiers that are potent inducers of cytokines, including IFN-alpha, TNF-alpha, IL-12, and IFN-gamma. Many of these cytokines can affect the acquired immune response. This study examines the effects of R-848 on aspects of acquired immunity, including immunoglobulin secretion, in vivo cytokine production, and Ag-specific T cell cytokine production. Results are compared with those of Th1 CpG ODN. R-848 and CpG ODN are effective at skewing immunity in the presence of Alum toward a Th1 Ab response (IgG2a) and away from a Th2 Ab response (IgE). R-848 and CpG ODN are also capable of initiating an immune response in the absence of additional adjuvant by specifically enhancing IgG2a levels. Both R-848 and imiquimod showed activity when given subcutaneously or orally, indicating that the compound mechanism was not through generation of a depot effect. Although CpG ODN behaves similarly to R-848, CpG ODN has a distinct cytokine profile, is more effective than R-848 when given with Alum in the priming dose, and is active only when given by the same route as the Ag. The mechanism of R-848's adjuvant activity is linked to cytokine production, where increases in IgG2a levels are associated with IFN-alpha, TNF-alpha, IL-12, and IFN-gamma induction, and decreases in IgE levels are associated with IFN-alpha and TNF-alpha. Imiquimod also enhances IgG2a production when given with Ag. The above results suggest that the imidazoquinolines R-848 and imiquimod may be attractive compounds for use as vaccine adjuvants and in inhibiting pathological responses mediated by Th2 cytokines.

Dendritic cell maturation and subsequent enhanced T-cell stimulation induced with the novel synthetic immune response modifier R-848.

Agents that enhance dendritic cell maturation can enhance T-cell activation and therefore may improve the efficiency of vaccines or improve cellular immunotherapy. Previously, we demonstrated that a novel low-molecular-weight synthetic immune response modifier, R-848, induces IL-12 and IFN-alpha secretion from monocytes and macrophages. Here we report that R-848 induces the maturation of human monocyte-derived dendritic cells. Characteristic of dendritic cell maturation, R-848 treatment induces cell surface expression of CD83 and increases cell surface expression of CD80, CD86, CD40, and HLA-DR. Additionally, R-848 induces cytokine (IL-6, IL-12, TNF-alpha, IFN-alpha) and chemokine (IL-8, MIP-1alpha, MCP-1) secretion from dendritic cells. Most significantly, R-848 enhances dendritic cell antigen presenting function, as measured by increased T-cell proliferation and T-cell cytokine secretion in both allogeneic and autologous T-cell systems. Consequently, low-molecular-weight synthetic molecules such as R-848 and its derivatives may be useful as vaccine adjuvants or as ex vivo stimulators of dendritic cells for cellular immunotherapy.