Novel, Meso-Substituted Cationic Porphyrin Molecule for Photo-Mediated Larval Control of the Dengue Vector Aedes aegypti

Dengue is a life-threatening viral disease of growing importance, transmitted by Aedes mosquito vectors. The control of mosquito larvae is crucial to contain or prevent disease outbreaks, and the discovery of new larvicides able to increase the efficacy and the flexibility of the vector control approach is highly desirable. Porphyrins are a class of molecules which generate reactive oxygen species if excited by visible light, thus inducing oxidative cell damage and cell death. In this study we aimed at assessing the potential of this photo-mediated cytotoxic mechanism to kill Aedes (Stegomyia) aegypti mosquito larvae. The selected porphyrin molecule, meso-tri(N-methylpyridyl),meso-mono(N-tetradecylpyridyl)porphine (C14 for simplicity), killed the larvae at doses lower than 1 µM, and at light intensities 50–100 times lower than those typical of natural sunlight, by damaging their intestinal tissues. The physicochemical properties of C14 make it easily adsorbed into organic material, and we exploited this feature to prepare an ‘insecticidal food’ which efficiently killed the larvae and remained active for at least 14 days after its dispersion in water. This study demonstrated that photo-sensitizing agents are promising tools for the development of new larvicides against mosquito vectors of dengue and other human and animal diseases

Sunlight-activatable porphyrin formulations as potential larviciding tool for the control of the Afrotropical malaria vector, Anopheles gambiae s.l.

Photolarvicidal formulations based on the use of a porphyrin photosensitizer associated to carriers palatable to mosquito larvae could represent a new approach for the development of larvicides suitable for the control of malaria vectors.\ud We demonstrated a high larvicidal efficacy of diet-porphyrin based formulations comprising meso-tri(N-methyl-pyridyl),mono(N-dodecyl-pyrydyl)porphine (known as C12 porphyrin) and ground cat food pellet (CF) or pollen granules (PO) as C12 carriers against Anopheles gambiae s.l. in semi-natural condition based on outdoor tray experiments conducted in Burkina Faso. Rapid larval mortality was obtained, in particular by a fine particles fraction from the C12-CF preparation (particles sizes < 160 μm). The efficacy of the various formulations was found to be influenced by particle sizes, type of formulation and breeding water type, rather than the mosquito species. The C12-CF-fine formulation caused 100% mortality after 3.5 h exposure to sunlight at all conditions, whereas C12-CF-coarse and C12-PO required more sunlight exposure time (8 to 20 h) to cause relevant larval mortality, which also varied with the type of water and to a lesser degree with the Anopheles species/population used. The formulations showed a limited residual activity. One day of pre-exposure to direct sunlight was sufficient to completely abrogate the photocidal activity of the C12-CF-coarse formulation and to decrease the mortality rate induced by C12-CF-fine and C12-PO to 27% and 33% respectively.\ud The evaluation of the effectiveness of the C12 porphyrin-diet formulations against larvae of An. gambiae mosquitoes in their natural habitats through controlled-field experiments showed that for an optimal use, such formulations preferably should be based on fine particle carriers measuring less than 160 μm. The finely formulated C12-diets (C12-CF-fine, C12-PO) demonstrated, within 24 h after treatment of these breeding habitats, 50 – 100% reduction in the density of immature anopheline (with variation according to the larval stages). Moreover, this evaluation showed that more formulation optimization studies are required for such larvicadal preparations to fulfill requirements for anopheline control.\ud Taking into account the antifeedant and insect growth regulatory properties of azadirachtin A, a secondary metabolite abundantly present in the seed kernel of neem (Azadirachtin indica), we investigated combinations of C12 porphyrin and neem products, for their capability to induce both rapid killing and delayed anti-larval effects on larvae of Anopheles gambiae mosquitoes. Alike the C12-loaded CF formulation, C12-loaded neem products,\ud particularly C12-loaded neem fruit (C12-NF) and C12-loaded neem leaves (C12-NL) yielded rapid killing, causing almost complete larval mortality 48 h after-treatment in spring water. This effect was found to be reduced in water samples from some natural breeding sites. However, a prolonged exposure allowed to achieve a delayed-effect after 4 – 5 days post-treatment, and larval mortalities of 70 – 100% were still observed in water types rich of soil particles (and probably biotic and organic matter). Upon a extended-exposure time of 9 days, C12-NF and the C12-loaded 10% NeemAzal®-treated CF (C12-NACF) yielded a residual activity in spring water, of about 75 – 100% larval mortality; while the neem-free C12-loaded product, C12-CF, showed only 21% larval mortality after the same time of extended-exposure.\ud The combination of C12 porphyrin with a neem product, in particular the C12-NF and C12-NACF formulations, showed an additive efficacy on larvae of Anopheles gambiae s.l. in this preliminary study. Further investigations in natural conditions of larval habitats should help to clarify the potential of such combinations for the development of a bio-larvicide approriate for malaria vector control

Residual activity of C14 solutions and formulates on <i>Ae. aegypti</i> larvae.

<p>Mortality was assessed after 12 h irradiation (1.0–4.0 mW/cm²).</p><p>*time elapsed between the preparation of the trays and the introduction of 3^rd–4^th instar larvae (n = 100 larvae; 3 replicates). During this period, trays were incubated in the climatic chamber (12 h photoperiod; 28±2°C; >90% RH). Numbers in parentheses indicate standard deviations.</p>†<p>trays contained 6 mg of untreated larval food in C14 porphyrin solutions at the indicated concentration.</p>‡<p>trays contained 6 mg of the indicated formulation in spring water.</p>§<p>one surviving larva was found in the tray. Such larvae were negative for C14 fluorescence at the microscope, therefore they hadn't fed during the experiment.</p

Survival of <i>Ae. aegypti</i> larvae exposed to C14 in the dark.

a<p>incubation was carried out with 5 µM C14 in the dark at 28±2°C; control larvae were incubated for 24 h.</p>b<p>surviving larvae at the end of the incubation period (n = 3 replicates of ∼50 larvae each). Numbers in parentheses indicate standard deviations.</p>c<p>Emerged adults/total pupae; pooled data from the three replications.</p>d<p>12 h-long irradiation (1.0–4.0 mW/cm²).</p

Adsorption and release dynamics of C14 on PFP (particle size 5–500 µm).

<p><b>A</b>: Residual concentration of C14 (5 µM) as function of incubation time at 28±2°C in the presence (▪) and in absence (•) of PFP (70 mg/500 ml C14 solution); <b>B</b>: stability of the C14-loaded PFP at various pH values.</p

Effect of concentration on the absorbance of C14 porphyrin solutions.

<p>Solutions were prepared in PBS. <b>A:</b> absorbance of solutions at the maximum of the Soret band (424 nm); <b>B:</b> absorbance at a wavelength characterized by a lower molar extinction coefficient (404 nm).</p

Median lethal concentrations (LC₅₀) of C14 porphyrin.

<p>C14 solutions at 7 increasing concentrations (range 0.03–4.3 µM) were incubated with 6 mg PFP at 28±2°C in the dark for 48 h. <i>Ae. aegypti</i> larvae (3^rd–early 4^th instar, n = 100, 3 replicates) were introduced 12 hours before the beginning of the irradiation (1.0–4.0 mW/cm²).</p

Efficiency of singlet oxygen generation by photoactivated C14 porphyrin.

<p>Effect of the irradiation time on the fluorescence properties of a DMA solution (initial absorbance around 1 at 380 nm) and porphyrin solution (initial absorbance around 0.4 at 420 nm) in N,N-dimethyl-formamide (DMF), which was exposed to white light (400–800 nm) at a fluence rate of 100 mW/cm². The spectra taken at 1, 3, 5, 10 and 15 s were overlapping, and the corresponding coloured lines have been omitted from the legend, for clarity.</p

Chemical structure of C14 porphyrin: meso-tri(N-methylpyridyl),meso-mono(N-tetradecylpyridyl)porphine.

<p>Chemical structure of C14 porphyrin: meso-tri(N-methylpyridyl),meso-mono(N-tetradecylpyridyl)porphine.</p