Infectivity of Plasmodium parasites to Aedes aegypti and Anopheles stephensi mosquitoes maintained on blood-free meals of SkitoSnack.

BACKGROUND: Aedes and Anopheles mosquitoes are responsible for tremendous global health burdens from their transmission of pathogens causing malaria, lymphatic filariasis, dengue, and yellow fever. Innovative vector control strategies will help to reduce the prevalence of these diseases. Mass rearing of mosquitoes for research and support of these strategies presently depends on meals of vertebrate blood, which is subject to acquisition, handling, and storage issues. Various blood-free replacements have been formulated for these mosquitoes, but none of these replacements are in wide use, and little is known about their potential impact on competence of the mosquitoes for Plasmodium infection. METHODS: Colonies of Aedes aegypti and Anopheles stephensi were continuously maintained on a blood-free replacement (SkitoSnack; SS) or bovine blood (BB) and monitored for engorgement and hatch rates. Infections of Ae. aegypti and An. stephensi were assessed with Plasmodium gallinaceum and P. falciparum, respectively. RESULTS: Replicate colonies of mosquitoes were maintained on BB or SS for 10 generations of Ae. aegypti and more than 63 generations of An. stephensi. The odds of engorgement by SS- relative to BB-maintained mosquitoes were higher for both Ae. aegypti (OR = 2.6, 95% CI 1.3-5.2) and An. stephensi (OR 2.7, 95% CI 1.4-5.5), while lower odds of hatching were found for eggs from the SS-maintained mosquitoes of both species (Ae. aegypti OR = 0.40, 95% CI 0.26-0.62; An. stephensi OR = 0.59, 95% CI 0.36-0.96). Oocyst counts were similar for P. gallinaceum infections of Ae. aegypti mosquitoes maintained on SS or BB (mean ratio = [mean on SS]/[mean on BB] = 1.11, 95% CI 0.85-1.49). Similar oocyst counts were also observed from the P. falciparum infections of SS- or BB-maintained An. stephensi (mean ratio = 0.76, 95% CI 0.44-1.37). The average counts of sporozoites/mosquito showed no evidence of reductions in the SS-maintained relative to BB-maintained mosquitoes of both species. CONCLUSIONS: Aedes aegypti and An. stephensi can be reliably maintained on SS over multiple generations and are as competent for Plasmodium infection as mosquitoes maintained on BB. Use of SS alleviates the need to acquire and preserve blood for mosquito husbandry and may support new initiatives in fundamental and applied research, including novel manipulations of midgut microbiota and factors important to the mosquito life cycle and pathogen susceptibility.

Aedes aegypti SGS1 is critical for Plasmodium gallinaceum infection of both the mosquito midgut and salivary glands.

BACKGROUND: The invasion of the mosquito salivary glands by Plasmodium sporozoites is a critical step that defines the success of malaria transmission and a detailed understanding of the molecules responsible for salivary gland invasion could be leveraged towards control of vector-borne pathogens. Antibodies directed against the mosquito salivary gland protein SGS1 have been shown to reduce Plasmodium gallinaceum sporozoite invasion of Aedes aegypti salivary glands, but the specific role of this protein in sporozoite invasion and in other stages of the Plasmodium life cycle remains unknown. METHODS: RNA interference and CRISPR/Cas9 were used to evaluate the role of A. aegypti SGS1 in the P. gallinaceum life cycle. RESULTS: Knockdown and knockout of SGS1 disrupted sporozoite invasion of the salivary gland. Interestingly, mosquitoes lacking SGS1 also displayed fewer oocysts. Proteomic analyses confirmed the abolishment of SGS1 in the salivary gland of SGS1 knockout mosquitoes and revealed that the C-terminus of the protein is absent in the salivary gland of control mosquitoes. In silico analyses indicated that SGS1 contains two potential internal cleavage sites and thus might generate three proteins. CONCLUSION: SGS1 facilitates, but is not essential for, invasion of A. aegypti salivary glands by P. gallinaceum and has a dual role as a facilitator of parasite development in the mosquito midgut. SGS1 could, therefore, be part of a strategy to decrease malaria transmission by the mosquito vector, for example in a transgenic mosquito that blocks its interaction with the parasite.

Net charge tuning modulates the antiplasmodial and anticancer properties of peptides derived from scorpion venom.

VmCT1, a linear helical antimicrobial peptide isolated from the venom of the scorpion Vaejovis mexicanus, displays broad spectrum antimicrobial activity against bacteria, fungi, and protozoa. Analogs derived from this peptide containing single Arg-substitutions have been shown to increase antimicrobial and antiparasitic activities against Trypanossoma cruzi. Here, we tested these analogs against malaria, an infectious disease caused by Plasmodium protozoa, and assessed their antitumoral properties. Specifically, we tested VmCT1 synthetic variants [Arg]3 -VmCT1-NH2 , [Arg]7 -VmCT1-NH2 , and [Arg]11 -VmCT1-NH2 , against Plasmodium gallinaceum sporozoites and MCF-7 mammary cancer cells. Our screen identified peptides [Arg]3 -VmCT1-NH2 and [Arg]7 -VmCT1-NH2 as potent antiplasmodial agents (IC50 of 0.57 and 0.51 µmol L-1 , respectively), whereas [Arg]11 -VmCT1-NH2 did not show activity against P. gallinaceum sporozoites. Interestingly, all peptides presented activity against MCF-7 and displayed lower cytotoxicity toward healthy cells. We demonstrate that increasing the net positive charge of VmCT1, through arginine substitutions, modulates the biological properties of this peptide family yielding novel antiplasmodial and antitumoral molecules.

Vector competence of Aedes albopictus (Skuse) and Aedes aegypti (Linnaeus) for Plasmodium gallinaceum infection and transmission.

Avian malaria caused by Plasmodium gallinaceum is an important mosquito-borne disease. Eradication of this disease remains problematic since its competent vectors are diverse and widely distributed across the globe. Several mosquito species were implicated as competent vectors for this parasite. However, studies on vector competence for P. gallinaceum remain limited. In this study, vector competence in the two most predominant mosquito vectors in tropical countries, Aedes albopictus and Ae. aegypti, was compared. In order to determine their infection rates, Ae. albopictus (>F10), Ae. aegypti (>F10), and Ae. aegypti ( F10) and Ae. aegypti (> F10) and 40-60% by infected Ae. aegypti (F10) and Ae. aegypti (>F10) were highly competent vectors for P. gallinaceum infections. These mosquitoes play a crucial role in the transmission cycle of this parasite in nature.

Primaquine-thiazolidinones block malaria transmission and development of the liver exoerythrocytic forms.

BACKGROUND: Primaquine is an anti-malarial used to prevent Plasmodium vivax relapses and malaria transmission. However, PQ metabolites cause haemolysis in patients deficient in the enzyme glucose-6-phosphate dehydrogenase (G6PD). Fifteen PQ-thiazolidinone derivatives, synthesized through one-post reactions from primaquine, arenealdehydes and mercaptoacetic acid, were evaluated in parallel in several biological assays, including ability to block malaria transmission to mosquitoes. RESULTS: All primaquine derivatives (PQ-TZs) exhibited lower cell toxicity than primaquine; none caused haemolysis to normal or G6PD-deficient human erythrocytes in vitro. Sera from mice pretreated with the test compounds thus assumed to have drug metabolites, caused no in vitro haemolysis of human erythrocytes, whereas sera from mice pretreated with primaquine did cause haemolysis. The ability of the PQ-TZs to block malaria transmission was evaluated based on the oocyst production and percentage of mosquitoes infected after a blood meal in drug pre-treated animals with experimental malaria caused by either Plasmodium gallinaceum or Plasmodium berghei; four and five PQ-TZs significantly inhibited sporogony in avian and in rodent malaria, respectively. Selected PQ-TZs were tested for their inhibitory activity on P. berghei liver stage development, in mice and in vitro, one compound (4m) caused a 3-day delay in the malaria pre-patent period. CONCLUSIONS: The compound 4m was the most promising, blocking malaria transmissions and reducing the number of exoerythrocytic forms of P. berghei (EEFs) in hepatoma cells in vitro and in mice in vivo. The same compound also caused a 3-day delay in the malaria pre-patent period.

Artesunate-tafenoquine combination therapy promotes clearance and abrogates transmission of the avian malaria parasite Plasmodium gallinaceum.

Clinical manifestations of malaria infection in vertebrate hosts arise from the multiplication of the asexual stage parasites in the blood, while the gametocytes are responsible for the transmission of the disease. Antimalarial drugs that target the blood stage parasites and transmissible gametocytes are rare, but are essentially needed for the effective control of malaria and for limiting the spread of resistance. Artemisinin and its derivatives are the current first-line antimalarials that are effective against the blood stage parasites and gametocytes, but resistance to artemisinin has now emerged and spread in various malaria endemic areas. Therefore, a novel antimalarial drug, or a new drug combination, is critically needed to overcome this problem. The objectives of this study were to evaluate the efficacy of a relatively new antimalarial compound, tafenoquine (TQ), and a combination of TQ and a low dose of artesunate (ATN) on the in vivo blood stage multiplication, gametocyte development and transmission of the avian malaria parasite Plasmodium gallinaceum to the vector Aedes aegypti. The results showed that a 5-d treatment with TQ alone was unable to clear the blood stage parasites, but was capable of reducing the mortality rate, while TQ monotherapy at a high dose of 30mg/kg was highly effective against the gametocytes and completely blocked the transmission of P. gallinaceum. In addition, the combination therapy of TQ+ATN completely cleared P. gallinaceum blood stages and sped up the gametocyte clearance from chickens, suggesting the synergistic effect of the two drugs. In conclusion, TQ is demonstrated to be effective for limiting avian malaria transmission and may be used in combination with a low dose of ATN for safe and effective treatment.

Endogenously-expressed NH2-terminus of circumsporozoite protein interferes with sporozoite invasion of mosquito salivary glands.

BACKGROUND: The circumsporozoite protein is the most abundant polypeptide expressed by sporozoites, the malaria parasite stage capable of infecting humans. Sporozoite invasion of mosquito salivary glands prior to transmission is likely mediated by a receptor/ligand-like interaction of the parasites with the target tissues, and the amino (NH2)-terminal portion of CSP is involved in this interaction but not the TSR region on the carboxyl (C)-terminus. Peptides based on the NH2-terminal domain could compete with the parasites for the salivary gland receptors and thus inhibit penetration. METHODS: Peptides based on the NH2-terminus and TSR domains of the CSP from avian or human malaria parasites, Plasmodium gallinaceum and Plasmodium falciparum, respectively, were expressed endogenously in mosquito haemolymph using a transient (Sindbis virus-mediated) or stable (piggyBac-mediated transgenesis) system. RESULTS: Transient endogenous expression of partial NH2-terminus peptide from P. falciparum CSP in P. gallinaceum-infected Aedes aegypti resulted in a reduced number of sporozoites in the salivary glands. When a transgenic approach was used to express a partial CSP NH2-terminal domain from P. gallinaceum the number of sporozoites in the salivary glands did not show a difference when compared to controls. However, a significant difference could be observed when mosquitoes with a lower infection were analysed. The same result could not be observed with mosquitoes endogenously expressing peptides based on the TSR domain from either P. gallinaceum or P. falciparum. CONCLUSION: These results support the conclusion that CSP partial NH2-terminal domain can be endogenously expressed to promote a competition for the receptor used by sporozoites to invade salivary glands, and they could be used to block this interaction and reduce parasite transmission. The same effect cannot be obtained with peptides based on the TSR domain.

Antiplasmodial activity study of angiotensin II via Ala scan analogs.

Angiotensin II (AII) as well as analog peptides shows antimalarial activity against Plasmodium gallinaceum and Plasmodium falciparum, but the exact mechanism of action is still unknown. This work presents the solid-phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide-capped derivative and the evaluation of the antiplasmodial activity of these peptides against mature P. gallinaceum sporozoites. The Ala screening data indicates that the replacement of either the Ile(5) or the His(6) residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala(6) ]-AII (79%), and [Ala(5) ]-AII (75%). Analogs [Ala(3) ]-AII, [Ala(1) ]-AII, and AII-NH2 showed antiplasmodial activity around 65%, whereas the activity of the [Ala(8) ]-AII, [Ala(7) ]-AII, [Ala(4) ]-AII, and [Ala(2) ]-AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a ß-fold conformation in different solutions. All AII analogs, except [Ala(4) ]-AII and [Ala(8) ]-AII, show contractile responses and interact with the AT1 receptor, [Ala(5) ]-AII and [Ala(6) ]-AII. In conclusion, this approach is helpful to understand the contribution of each amino acid residue to the bioactivity of AII, opening new perspectives toward the design of new sporozoiticidal compounds.

A study of the anti-plasmodium activity of angiotensin II analogs.

Controlling the dissemination of malaria requires the development of new drugs against its etiological agent, a protozoan of the Plasmodium genus. Angiotensin II and its analog peptides exhibit activity against the development of immature and mature sporozoites of Plasmodium gallinaceum. In this study, we report the synthesis and characterization of angiotensin II linear and cyclic analogs with anti-plasmodium activity. The peptides were synthesized by a conventional solid-phase method on Merrifield's resin using the t-Boc strategy, purified by RP-HPLC and characterized by liquid chromatography/ESI (+) MS (LC-ESI(+)/MS), amino acid analysis, and capillary electrophoresis. Anti-plasmodium activity was measured in vitro by fluorescence microscopy using propidium iodine uptake as an indicator of cellular damage. The activities of the linear and cyclic peptides are not significantly different (p < 0.05). Kinetics studies indicate that the effects of these peptides on plasmodium viability overtime exhibit a sigmoidal profile and that the system stabilizes after a period of 1 h for all peptides examined. The results were rationalized by partial least-square analysis, assessing the position-wise contribution of each amino acid. The highest contribution of polar amino acids and a Lys residue proximal to the C-terminus, as well as that of hydrophobic amino acids in the N-terminus, suggests that the mechanism underlying the anti-malarial activity of these peptides is attributed to its amphiphilic character.

Some newer knowledge in parasitology: a review

A review of the literature on parasitology, which we did not have the opportunity to get acquainted with during the Japanese occupation but which was made accessible to us after liberation, has disclosed a number of interesting facts and additional knowledge which are here presented for whatever benefit may be derived by the medical practitioners, medical students, and scientific investigators in this country. It includes new knowledge on: (1) the treatment of clinical malaria with bigger doses of atabrine-28 to 42 tablets of 0.10 Gm. for one course of 8 days instead of only 15 tablets in 5 days for adults; (2) the definite preference of the American forces for atabrine over quinine in both suppressive and clinical treatments of malaria; (3) the negative action of the wonder drug penicillin against induced or inoculation malaria in man; (4) the good effect of tyrothricin, another antibiotic, in experimental Plasmodium gallinaceum infection in chickens when given intravenously; (5) the most recently announced drugs such as Paludrine and SN 7618 against malaria: (6) the use of newer sulfonamides like sulfadiazine and sulfapyrazine in the eradication of the exo-erythrocytic stages of Plasmodium gallinaceum and the failure of the same drug against the same stages of Plasmodium elongatum; (7) the present status of immunity in malaria and its mechanism of production supporting the combined operation of the phagocytic and humoral theories; (8) the more recent attempt at active immunization of ducklings from Plasmodium lophurae infection with the use of plasmodial residues mixed with Staphylococcus toxoid, which may pave the way for the elaboration of a vaccine against human malaria; (9) the exo-erythrocytic cycle in malaria, with special emphasis on its present status as applied to the human species; (10) the use of DDT, the wonder insecticide of World War II in malaria control; (11) the new acid-ether technic of fecal examination for Schistosoma japonicum eggs; (12) the effectiveness of one part per million (1 P.P.M.) residual chlorine against Schistosoma japonicum cercariae when applied to infected waters; (13) the early manifestations of filariasis bancrofti and the use of the intra-dermal test for early diagnosis; (14) the improvement suggested in enhancing the positive diagnosis of amoebiasis by fixation of fecal smears at the bedside and the permanent staining afterwards of the same in the laboratory; (15) the present status of complement-fixation test in the diagnosis of amoebiasis; (16) the use of diodoquin as an anti-amoebic drug; (17) a report of vaginitis of amoebic etiology; (18) the possible pathogenicity of Dientamoeba fragilis; (19) some unusual radislogical findings in giardiasis which may prove confusing at times in the diagnosis of peptic ulcer; and(20) the previously unrecognized disease entity of man called human toxoplasmosis. (Summary)

COSTS AND BENEFITS OF MOSQUITO REFRACTORINESS TO MALARIA PARASITES: IMPLICATIONS FOR GENETIC VARIABILITY OF MOSQUITOES AND GENETIC CONTROL OF MALARIA.

The problem of fitness costs associated with host resistance to parasitism is related to the evolution of parasite virulence, population genetic diversity and the dynamics of host-parasite relationships, and proposed strategies for disease control through the genetic manipulation of mosquito vectors. Two Aedes aegypti populations, refractory and susceptible to Plasmodium gallinaceum, were previously selected from the Moyo-In-Dry strain (MOYO) through inbreeding (F = 0.5). Reproductive success and survivorship of the two populations were compared, and the influence of the parasite on mosquito fitness also was evaluated. Fitness components studied include fecundity, adult survivorship and egg-to-adult developmental time, blood-meal size, and adult body size. The refractory population has a significantly shorter egg-to-adult developmental time and a smaller body size, takes a smaller blood meal, and subsequently lays fewer eggs than the susceptible population. The mean longevity of the refractory population is significantly shorter than the susceptible population. Exposure to the parasite exhibited little effect on the survivorship and fecundity of either population. Several factors may contribute to the lower fitness of the refractory population, including founder effect, inbreeding depression, the effect of other uncharacterized genes linked to genes conferring refractoriness, and pleiotropic effects associated with these genes. The results are discussed in relation to the genetic diversity of natural mosquito populations and their implications for the genetic control of malaria.