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Avian haemosporidian parasites are spread worldwide and pose a threat to their hosts occasionally. A complete life cycle of these parasites requires two hosts: vertebrate and invertebrate (a blood-sucking insect that acts as a vector). In this study, we tested wild-caught mosquitoes for haemosporidian infections. Mosquitoes were collected (2021-2023) in several localities in Lithuania using a sweeping net and a CDC trap baited with CO 2 , morphologically identified, and preparations of salivary glands were prepared (from females collected in 2022-2023). 2093 DNA samples from either individual after dissection (1675) or pools (418 pools/1145 individuals) of female mosquito's abdomens were screened using PCR for the detection of haemosporidian parasite DNA. Salivary gland preparations were analyzed microscopically from each PCR-positive mosquito caught in 2022 and 2023. The average prevalence of haemosporidian parasites for all analyzed samples was 2.0 % and varied between 0.6 % (2021) and 3.5 % (2022). DNA of Plasmodium ashfordi (cytochrome b genetic lineage pGRW02), P. circumflexum (pTURDUS1), P. homonucleophilum (pSW2), P. matutinum (pLINN1), P. vaughani (pSYAT05), Haemoproteus brachiatus (hLK03), H. majoris (hWW2), and H. minutus (hTUPHI01) were detected in mosquitoes. Coquilletidia richiardii (3.5 %) and Culex pipiens (2.9 %) were mosquito species with the highest prevalence of haemosporidian parasite DNA detected. Mixed infections were detected in 16 mosquitoes. In one of the samples, sporozoites of P. matutinum (pLINN1) were found in the salivary gland preparation of Culex pipiens, confirming this mosquito species as a competent vector of Plasmodium matutinum and adding it to the list of the natural vectors of this avian parasite., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Malaria is initiated when infected anopheline mosquitoes inoculate sporozoites as they probe for blood. It is thought that all infected mosquitoes are equivalent in terms of their infectious potential, with parasite burden having no role in transmission success. In this study, using mosquitoes harboring the entire range of salivary gland sporozoite loads observed in the field, we demonstrate a strong and highly significant correlation between mosquito parasite burden and inoculum size. We then link the inoculum data to oocyst counts, the most commonly-used metric to assess mosquito infection in the field, and determine the efficiency with which oocyst sporozoites enter mosquito salivary glands. Taken together our data support the conclusion that mosquitoes with higher parasite burdens are more likely to initiate infection and contribute to onward transmission. Overall these data may account for some of the unexplained heterogeneity in transmission and enable more precise benchmarks for transmission-blocking interventions., (© 2024. The Author(s).)

Background: Malaria remains a major public health problem in the Republic of Congo, with Plasmodium falciparum being the deadliest species of Plasmodium in humans. Vector transmission of malaria is poorly studied in the country and no previous report compared rural and urban data. This study aimed to determine the Anopheles fauna and the entomological indices of malaria transmission in the rural and urban areas in the south of Brazzaville, and beyond., Methods: Indoor household mosquitoes capture using electric aspirator was performed in rural and urban areas during raining and dry seasons in 2021. The identification of Anopheles species was done using binocular magnifier and nested-PCR. TaqMan and nested-PCR were used to detect the Plasmodium species in the head/thorax and abdomens of Anopheles. Some entomological indices including the sporozoite infection rate, the entomological inoculation rate and the man biting rate were estimated., Results: A total of 699 Anopheles mosquitoes were collected: Anopheles gambiae sensu lato (s.l.) (90.7%), Anopheles funestus s.l. (6.9%), and Anopheles moucheti (2.4%). Three species of An. gambiae s.l. were identified including Anopheles gambiae sensu stricto (78.9%), Anopheles coluzzii (15.4%) and Anopheles arabiensis (5.7%). The overall sporozoite infection rate was 22.3% with a predominance of Plasmodium falciparum, followed by Plasmodium malariae and Plasmodium ovale. Anopheles aggressiveness rate was higher in households from rural area (1.1 bites/night) compared to that from urban area (0.8 ib/p/n). The overall entomological inoculation rate was 0.13 ib/p/n. This index was 0.17 ib/p/n and 0.092 ib/p/n in rural and in urban area, respectively, and was similar during the dry (0.18 ib/p/n) and rainy (0.14 ib/p/n) seasons., Conclusion: These findings highlight that malaria transmission remains high in rural and urban area in the south of Republic of Congo despite the ongoing control efforts, thereby indicating the need for more robust interventions., (© 2024. The Author(s).)

Malaria and lymphatic filariasis (LF) are two serious public health challenges in sub-Saharan Africa, and both diseases are transmitted by Anopheles mosquitoes. Successful control of both diseases requires detailed information on transmission dynamics; thus, this study investigated malaria and LF transmission indices in two (2) communities (Jidawa and Kargo) in North-West Nigeria. Anopheles mosquitoes were sampled from twenty-five (25) randomly selected houses from each of the two communities using pyrethrum spray collection (PSC). The samples were identified morphologically and molecularly characterised using polymerase chain reaction (PCR). Human biting rate (HBR), indoor resting density (IRD), sporozoite rate (SR) and entomological inoculation rate (EIR) were calculated using standard formulae. The thorax region of the collected samples were dissected and smeared; then, Plasmodium and Wuchereria bancrofti parasites were identified using microscopy. A total of 2417 Anopheles mosquitoes were collected, and all were identified morphologically as An. gambiae s.l. Further molecular identification of sibling species revealed that An. gambiae and An. arabiensis were the only sibling species present. A total of 818 Anopheles mosquitoes were screened for Plasmodium and Wuchereria bancrofti parasites. A total of 180 samples were positive for Plasmodium parasites (Jidawa = 151; Kargo = 29), and none was positive for W. bancrofti (0%). Result of entomological indices for malaria transmission showed that indoor resting density was higher in Jidawa (10 mosquitoes/room/night) while human biting rate (2.07 bites/person/night), sporozoite rate (29.3%) and entomological inoculation rate (0.61) were higher in Kargo. In total, 35.2% of the samples were blood-fed while 67.4% were parous. There is active transmission of malaria in the two communities but not LF, suggesting the effectiveness of mass drug administration for LF. Concerted efforts should be focused on malaria control as transmission of the disease persists., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Adult mosquitoes of the genus Anopheles are important vectors of Plasmodium parasites, causative agents of malaria. The aim of this review was to synthesize the overall and species-specific proportion of Anopheles species infected with sporozoites and their geographical distribution in the last 2 decades (2001-2021)., Methods: A comprehensive search was conducted using databases (PubMed, Google Scholar, Science Direct, Scopus, African Journals OnLine) and manual Google search between January 1 and February 15, 2022. Original articles describing work conducted in Ethiopia, published in English and reporting infection status, were included in the review. All the required data were extracted using a standardized data extraction form, imported to SPSS-24, and analyzed accordingly. The quality of each original study was assessed using a quality assessment tool adapted from the Joanna Briggs Institute critical appraisal checklist. This study was registered on PROSPERO (International Prospective Register of Systematic Reviews; registration no. CRD42022299078)., Results: A search for published articles produced a total of 3086 articles, of which 34 met the inclusion criteria. Data on mosquito surveillance revealed that a total of 129,410 anophelines comprising 25 species were captured, of which 48,365 comprising 21 species were tested for sporozoites. Anopheles arabiensis was the dominant species followed by An. pharoensis and An. coustani complex. The overall proportion infected with sporozoites over 21 years was 0.87%. Individual proportions included Anopheles arabiensis (1.09), An. pharoensis (0.79), An. coustani complex (0.13), An. funestus (2.71), An. demeilloni (0.31), An. stephensi (0.70), and An. cinereus (0.73). Plasmodium falciparum sporozoites accounted 79.2% of Plasmodium species. Mixed infection of Plasmodium vivax and P. falciparum was only reported from one An. arabiensis sample., Conclusions: Anopheles arebiensis was the dominant malaria vector over the years, with the highest sporozoite infection proportion of 2.85% and an average of 0.90% over the years. Other species contributing to malaria transmission in the area were An. pharoensis, An. coustani complex, An. funestus, An. stephensi, and An. coustani. The emergence of new vector species, in particular An. stephensi, is particularly concerning and should be investigated further., (© 2023. The Author(s).)

Background: The extrinsic incubation period (EIP), defined as the time it takes for malaria parasites in a mosquito to become infectious to a vertebrate host, is one of the most influential parameters for malaria transmission but remains poorly understood. The EIP is usually estimated by quantifying salivary gland sporozoites in subsets of mosquitoes, which requires terminal sampling. However, assays that allow repeated sampling of individual mosquitoes over time could provide better resolution of the EIP., Methods: We tested a non-destructive assay to quantify sporozoites of two rodent malaria species, Plasmodium chabaudi and Plasmodium berghei, expelled throughout 24-h windows, from sugar-soaked feeding substrates using quantitative-PCR., Results: The assay is able to quantify sporozoites from sugar-soaked feeding substrates, but the prevalence of parasite-positive substrates was low. Various methods were attempted to increase the detection of expelled parasites (e.g. running additional technical replicates; using groups rather than individual mosquitoes), but these did not increase the detection rate, suggesting that expulsion of sporozoites is variable and infrequent., Conclusions: We reveal successful detection of expelled sporozoites from sugar-soaked feeding substrates. However, investigations of the biological causes underlying the low detection rate of sporozoites (e.g. mosquito feeding behaviour, frequency of sporozoite expulsion or sporozoite clumping) are needed to maximise the utility of using non-destructive assays to quantify sporozoite dynamics. Increasing detection rates will facilitate the detailed investigation on infection dynamics within mosquitoes, which is necessary to explain the highly variable EIP of Plasmodium and to improve understanding of malaria transmission dynamics., (© 2023. The Author(s).)

Attenuated plasmodial sporozoite-induced immune response includes intrahepatic nitric oxide (NO) production, which promotes apoptosis of infected hepatocytes and consequent parasite clearance. NO in excess reacts with superoxide, forming peroxynitrite, a powerful cytotoxic agent. Here, I suggest that peroxynitrite proapoptotic action may contribute to the attenuated malarial sporozoite-mediated protection., Competing Interests: Declaration of interests Although I am currently employed by Nykode Therapeutics ASA, the opinions and ideas stated in this article are solely mine and do not reflect the company’s position on the issues discussed., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The competence of insect vectors to transmit diseases plays a key role in host-parasite interactions and in the dynamics of avian malaria and other haemosporidian infections (Apicomplexa, Haemosporida). However, the presence of parasite DNA in the body of blood-sucking insects does not always constitute evidence for their competence as vectors. In this study, we investigate the susceptibility of wild-caught mosquitoes (Culex spp.) to complete sporogony of Plasmodium relictum (cyt b lineage SGS1) isolated from great tits (Parus major L., 1758). Adult female mosquitoes were collected with a CO 2 bait trap overnight. A set of 50 mosquitoes was allowed to feed for 3 h at night on a single great tit infected with P. relictum. This trial was repeated on 6 different birds. The bloodfed mosquitoes that survived (n = 68) were dissected within 1-2 days (for ookinetes, n = 10) and 10-33 days post infection (for oocysts and sporozoites, n = 58) in order to confirm the respective parasite stages in their organs. The experiment confirmed the successful development of P. relictum (cyt b lineage SGS1) to the stage of sporozoites in Culex pipiens L., 1758 (n = 27) and in Culex modestus (n = 2). Our study provides the first evidence that C. modestus is a competent vector of P. relictum isolated from great tits, suggesting that this mosquito species could also play a role in the natural transmission of avian malaria., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Plasmodium sporozoites actively migrate in the dermis and enter blood vessels to infect the liver. Despite their importance for malaria infection, little is known about these cutaneous processes. We combine intravital imaging in a rodent malaria model and statistical methods to unveil the parasite strategy to reach the bloodstream. We determine that sporozoites display a high-motility mode with a superdiffusive Lévy-like pattern known to optimize the location of scarce targets. When encountering blood vessels, sporozoites frequently switch to a subdiffusive low-motility behavior associated with probing for intravasation hotspots, marked by the presence of pericytes. Hence, sporozoites present anomalous diffusive motility, alternating between superdiffusive tissue exploration and subdiffusive local vessel exploitation, thus optimizing the sequential tasks of seeking blood vessels and pericyte-associated sites of privileged intravasation., (© 2023. The Author(s).)

Productive invasion of hepatocytes by Plasmodium sporozoites is a key step of infection. The parasites traverse hepatocytes before targeting one of them to form a parasitophorous vacuole for parasite expansion. Schepis et al. show the induction of membrane ruffling via host Rho GTPases by Plasmodium sporozoites facilitating productive invasion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Plasmodium sporozoites travel a long way from the site where they are released by a mosquito bite to the liver, where they infect hepatocytes and develop into erythrocyte-invasive forms. The success of this infection depends on the ability of the sporozoites to correctly recognize the hepatocyte as a target and change their behavior from migration to infection. However, how this change is accomplished remains incompletely understood. In this paper, we report that 6-cysteine protein family members expressed in sporozoites including B9 are responsible for this ability. Experiments on parasites using double knockouts of B9 and SPECT2, which is essential for sporozoite to migrate through the hepatocyte, showed that the parasites lacked the capacity to stop migration. This finding suggests that interactions between these parasite proteins and hepatocyte-specific cell surface ligands mediate correct recognition of hepatocytes by sporozoites, which is an essential step in malaria transmission to humans., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Plasmodium parasites have a complex life cycle alternating between a mosquito and a vertebrate host. Following the bite of an Anopheles female mosquito, Plasmodium sporozoites are transmitted from the skin to the liver; their first place of replication within the host. Successfully invaded sporozoites undergo a massive replication and growth involving asynchronous DNA replication and division that results in the generation of tens of thousands or even hundreds of thousands of merozoites depending on the Plasmodium species. The generation of a high number of daughter parasites requires biogenesis and segregation of organelles to finally reach a relatively synchronous cytokinesis event. At the end of liver stage (LS) development, merozoites are packed into merosomes and released into the bloodstream. They are then liberated and infect red blood cells to again produce merozoites by schizogony for the erythrocytic stage of the life cycle. Although parasite LS and asexual blood stage (ABS) differ in many respects, important similarities exist between the two. This review focuses on the cell division of Plasmodium parasite LS in comparison with other life cycle stages especially the parasite blood stage., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Roques et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Early Plasmodium falciparum and P. vivax infection requires parasite replication within host hepatocytes, referred to as liver stage (LS). However, limited understanding of infection dynamics in human LS exists due to species-specificity challenges. Reported here is a reproducible, easy-to-manipulate, and moderate-cost in vivo model to study human Plasmodium LS in mice; the ectopic huLiver model. Ectopic huLiver tumors were generated through subcutaneous injection of the HC-04 cell line and shown to be infectible by both freshly dissected sporozoites and through the bite of infected mosquitoes. Evidence for complete LS development was supported by the transition to blood-stage infection in mice engrafted with human erythrocytes. Additionally, this model was successfully evaluated for its utility in testing antimalarial therapeutics, as supported by primaquine acting as a causal prophylactic against P. falciparum. Presented here is a new platform for the study of human Plasmodium infection with the potential to aid in drug discovery., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Malaria is caused by the unicellular parasite Plasmodium which is transmitted to humans through the bite of infected female Anopheles mosquitoes. To initiate sexual reproduction and to infect the midgut of the mosquito, Plasmodium gametocytes are able to recognize the intestinal environment after being ingested during blood feeding. A shift in temperature, pH change and the presence of the insect-specific compound xanthurenic acid have been shown to be important stimuli perceived by gametocytes to become activated and proceed to sexual reproduction. Here we report that the salivary protein Saglin, previously proposed to be a receptor for the recognition of salivary glands by sporozoites, facilitates Plasmodium colonization of the mosquito midgut, but does not contribute to salivary gland invasion. In mosquito mutants lacking Saglin, Plasmodium infection of Anopheles females is reduced, resulting in impaired transmission of sporozoites at low infection densities. Interestingly, Saglin can be detected in high amounts in the midgut of mosquitoes after blood ingestion, possibly indicating a previously unknown host-pathogen interaction between Saglin and midgut stages of Plasmodium. Furthermore, we were able to show that saglin deletion has no fitness cost in laboratory conditions, suggesting this gene would be an interesting target for gene drive approaches., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

In 1880, Laveran observed the causative agent of malaria. As early as 1884, he considered that mosquitoes could be responsible for the transmission of haematozoa, a hypothesis which resulted from the observation and reflection of an informed hygienist. But, as Laveran himself said, "the opinion that I defended was considered by most observers to be highly unlikely".Nearly 15 years after the discovery of the haematozoan, the elucidation of the mechanism of transmission still proved difficult to establish. A link with the existence of swamps had been established a long time before, but the true mode of transmission remained a mystery until the end of the 19th century. The implication, by Manson in 1877, of mosquitoes in the cycle of the Bancroftian filaria, then other observations of the same order, ended up attracting the attention of malariologists. Laveran himself was quickly convinced of the role of mosquitoes in carrying out the natural cycle and propagating Plasmodium , but this theory had as many detractors as supporters.In 1897, Ross showed the presence of oocysts on the stomach of mosquitoes previously gorged on a malaria patient, then in 1898, of sporozoites of bird plasmodia in mosquitoes. He was convinced that, through their bite, these insects were responsible for the transmission of human malaria agents, without being able to prove it. The results obtained by Ross were immediately confirmed in Italy by Grassi and his collaborators who, in November 1898, described the stages of Plasmodium in man and, through various experiments carried out in collaboration with British researchers, showed the role of Anopheles , a result far from being accepted by all. Skepticism persisted for a long time.An excellent protozoologist, Laveran was not an entomologist. He was however among the first defenders of the anopheline theory. He worked extensively on establishing the relationships between Anopheles mosquitoes and malaria and took a close interest in the environmental conditions of the transmission. In his mind, malaria fever should henceforth be classified as a preventable disease. An era of hope thus dawned: malaria prophylaxis, based on fight against mosquitoes, could begin., (Copyright © 2023 SFMTSI.)

The liver is the first destination of malaria parasites in humans. After reaching the liver by the blood stream, Plasmodium sporozoites cross the liver sinusoid epithelium, enter and exit several hepatocytes, and eventually invade a final hepatocyte host cell. At present, the mechanism of hepatocyte invasion is only partially understood, presenting a key research gap with opportunities for the development of new therapeutics. Recently, human EphA2, a membrane-bound receptor tyrosine kinase, was implicated in hepatocyte infection by the human malaria parasite Plasmodium falciparum and the rodent parasite Plasmodium yoelii, but its role is not known for Plasmodium vivax, a major human parasite whose liver infection poses a specific challenge for malaria treatment and elimination. In this study, the role of EphA2 in P. vivax infection was investigated. It was found that surface expression of several recombinant fragments of EphA2 enhanced the parasite infection rate, thus establishing its role in P. vivax infection. Furthermore, a new permanent cell line (EphA2Extra-HC04) expressing the whole extracellular domain of EphA2 was generated. This cell line supports a higher rate of P. vivax infection and is a valuable tool for P. vivax liver-stage research., (© 2022. The Author(s).)

The complex Plasmodium life cycle offers different vaccine approaches with distinct parasitological and clinical effects. The approaches and their rationales were established decades ago: vaccines targeting pre-erythrocytic (sporozoite and liver-stage) parasites prevent infection, those to blood-stage parasites reduce disease, and those to sexual-stage parasites or mosquito vector reduce transmission and eliminate malaria through herd immunity. The pre-erythrocytic RTS,S vaccine (Mosquirix, GlaskoSmithKline (GSK)), recommended by WHO in 2021, reduces clinical malaria in children. Knowledge of parasite biology, host-parasite interactions, and immune mechanisms is informing new concepts to improve on RTS,S and to target other parasite stages. This review emphasizes vaccine approaches and candidates currently in the clinic or likely to enter clinical testing soon., (Copyright © 2022. Published by Elsevier Ltd.)

Mosquito saliva is a vehicle for the transmission of vector borne pathogens such as Plasmodium parasites and different arboviruses. Despite the key role of the salivary glands in the process of disease transmission, knowledge of host-pathogen interactions taking place within this organ is very limited. To improve the experimental tractability of the salivary glands, we have generated fluorescent reporter lines in the African malaria mosquito Anopheles coluzzii using the salivary gland-specific promoters of the anopheline antiplatelet protein (AAPP), the triple functional domain protein (TRIO) and saglin (SAG) coding genes. Promoter activity was specifically observed in the distal-lateral lobes or in the median lobe of the salivary glands. Besides a comparison of the expression patterns of the selected promoters, the fluorescent probes allowed us to evaluate the inducibility of the selected promoters upon blood feeding and to measure intracellular redox changes. We also combined the aapp-DsRed fluorescent reporter line with a pigmentation-deficient yellow(-) mosquito mutant to assess the feasibility of in vivo microscopy of parasitized salivary glands. This combination allowed locating the salivary gland through the cuticle and imaging of individual sporozoites in vivo, which facilitates live imaging studies of salivary gland colonization by Plasmodium sporozoites., Competing Interests: The authors declare that they have no conflict of interest.

Malaria is the cause of 600.000 deaths annually. However, these deaths represent only a tiny fraction of total malaria cases. Repeated natural infections with the causative agent, Plasmodium sp. parasites, induce protection from severe disease but not sterile immunity. Thus, immunity to Plasmodium is incomplete. Conversely, immunization with attenuated sporozoite stage parasites can induce sterile immunity albeit after multiple vaccinations. These different outcomes are likely to be influenced strongly by the innate immune response to different stages of the parasite lifecycle. Even small numbers of sporozoites can induce a robust proinflammatory type I interferon response, which is believed to be driven by the sensing of parasite RNA. Moreover, induction of innate like gamma-delta cells contributes to the development of adaptive immune responses. Conversely, while blood stage parasites can induce a strong proinflammatory response, regulatory mechanisms are also triggered. In agreement with this, intact parasites are relatively weakly sensed by innate immune cells, but isolated parasite molecules, notably DNA and RNA can induce strong responses. Thus, the innate response to Plasmodium parasite likely represents a trade-off between strong pro-inflammatory responses that may potentiate immunity and regulatory processes that protect the host from cytokine storms that can induce life threatening illness., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pohl and Cockburn.)

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8 + and gamma delta T cells (γδ T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8 + T, γδ T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

One hundred and twenty five years ago, in August 1897, Ronald Ross discovered forms of the malaria parasite in the gut of two mosquitoes. Shortly afterwards, Giovanni Battista Grassi established how the transmissive forms of the parasite, sporozoites, develop in these Plasmodium oocysts. Today, we still understand surprisingly little about the molecular processes governing oocyst biology., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Malaria is a devastating disease impacting over half of the world's population. Plasmodium parasites that cause malaria undergo obligatory development and replication in hepatocytes before infecting red blood cells and initiating clinical disease. While type I interferons (IFNs) are known to facilitate innate immune control to Plasmodium in the liver, how they do so has remained unresolved, precluding the manipulation of such responses to combat malaria. Utilizing transcriptomics, infection studies, and a transgenic Plasmodium strain that exports and traffics Cre recombinase, we show that direct type I IFN signaling in Plasmodium-infected hepatocytes is necessary to control malaria. We also show that the majority of infected hepatocytes naturally eliminate Plasmodium infection, revealing the potential existence of anti-malarial cell-autonomous immune responses in such hepatocytes. These discoveries challenge the existing paradigms in Plasmodium immunobiology and are expected to inspire anti-malarial drugs and vaccine strategies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the last few decades, considerable efforts have been made toward the development of efficient vaccines against malaria. Whole-sporozoite (Wsp) vaccines, which induce efficient immune responses against the pre-erythrocytic (PE) stages (sporozoites and liver forms) of Plasmodium parasites, the causative agents of malaria, are among the most promising immunization strategies tested until present. Several Wsp PE vaccination approaches are currently under evaluation in the clinic, including radiation- or genetically-attenuated Plasmodium sporozoites, live parasites combined with chemoprophylaxis, or genetically modified rodent Plasmodium parasites. In addition to the assessment of their protective efficacy, clinical trials of Wsp PE vaccine candidates inevitably involve the thorough investigation of the immune responses elicited by vaccination, as well as the identification of correlates of protection. Here, we review the main methodologies employed to dissect the humoral and cellular immune responses observed in the context of Wsp PE vaccine clinical trials and discuss future strategies to further deepen the knowledge generated by these studies, providing a toolbox for the in-depth analysis of vaccine-induced immunogenicity., (© 2021 Federation of European Biochemical Societies.)

In mammals, the serine protease plasmin degrades extracellular proteins during blood clot removal, tissue remodeling, and cell migration. The zymogen plasminogen is activated into plasmin by two serine proteases: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), a process regulated by plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor that specifically inhibits tPA and uPA. Plasmodium gametes and sporozoites use tPA and uPA to activate plasminogen and parasite-bound plasmin degrades extracellular matrices, facilitating parasite motility in the mosquito and the mammalian host. Furthermore, inhibition of plasminogen activation by PAI-1 strongly blocks infection in both hosts. To block parasite utilization of plasmin, we engineered Anopheles stephensi transgenic mosquitoes constitutively secreting human PAI-1 (huPAI-1) in the midgut lumen, in the saliva, or both. Mosquitoes expressing huPAI-1 strongly reduced rodent and human Plasmodium parasite transmission to mosquitoes, showing that co-opting plasmin for mosquito infection is a conserved mechanism among Plasmodium species. huPAI-1 expression in saliva induced salivary gland deformation which affects sporozoite invasion and P. berghei transmission to mice, resulting in significant levels of protection from malaria. Targeting the interaction of malaria parasites with the fibrinolytic system using genetically engineered mosquitoes could be developed as an intervention to control malaria transmission., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

During co-evolution Plasmodium parasites and vertebrates went through a process of selection resulting in defined and preferred parasite-host combinations. As such, Plasmodium falciparum (Pf ) sporozoites can infect human hepatocytes while seemingly incompatible with host cellular machinery of other species. The compatibility between parasite invasion ligands and their respective human hepatocyte receptors plays a key role in Pf host selectivity. However, it is unclear whether the ability of Pf sporozoites to mature in cross-species infection also plays a role in host tropism. Here we used fresh hepatocytes isolated from porcine livers to study permissiveness to Pf sporozoite invasion and development. We monitored intra-hepatic development via immunofluorescence using anti-HSP70, MSP1, EXP1, and EXP2 antibodies. Our data shows that Pf sporozoites can invade non-human hepatocytes and undergo partial maturation with a significant decrease in schizont numbers between day three and day five. A possible explanation is that Pf sporozoites fail to form a parasitophorous vacuolar membrane (PVM) during invasion. Indeed, the observed aberrant EXP1 and EXP2 staining supports the presence of an atypical PVM. Functions of the PVM include the transport of nutrients, export of waste, and offering a protective barrier against intracellular host effectors. Therefore, an atypical PVM likely results in deficiencies that may detrimentally impact parasite development at multiple levels. In summary, despite successful invasion of porcine hepatocytes, Pf development arrests at mid-stage, possibly due to an inability to mobilize critical nutrients across the PVM. These findings underscore the potential of a porcine liver model for understanding the importance of host factors required for Pf mid-liver stage development., Competing Interests: Author RS was employed by company TropIQ Health Sciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van der Boor, van Gemert, Hanssen, van Waardenburg, McCall, Bousema, de Wilt, Sauerwein and Yang.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Upon transmission to the human host, Plasmodium sporozoites exit the skin, are taken up by the blood stream, and then travel to the liver where they infect and significantly modify a single hepatocyte. Low infection rates within the liver have made proteomic studies of infected hepatocytes challenging, particularly in vivo , and existing studies have been largely unable to consider how protein and phosphoprotein differences are altered at different spatial locations within the heterogeneous liver. Using digital spatial profiling, we characterized changes in host signaling during Plasmodium yoelii infection in vivo without disrupting the liver tissue. Moreover, we measured alterations in protein expression around infected hepatocytes and identified a subset of CD163 + Kupffer cells that migrate towards infected cells during infection. These data offer the first insight into the heterogeneous microenvironment that surrounds the infected hepatocyte and provide insights into how the parasite may alter its milieu to influence its survival and modulate immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Glennon, Tongogara, Primavera, Reeder, Wei and Kaushansky.)

Vector-borne protozoan parasites such as Plasmodium spp. Leishmania spp. and Trypanosoma brucei are responsible for several serious diseases. Significant advances in parasitology have been made using rodent models combined with live imaging techniques, including whole-mouse bioluminescence imaging (BLI). This technique has been applied to investigate parasite dissemination, infectivity, and growth. It has also been used in drug and vaccine testing. This chapter focuses on the methods that utilize whole-mouse BLI to (i) evaluate the homing and infectivity of Plasmodium berghei sporozoites; (ii) conduct in vivo testing of promising chemical entities against Leishmania infantum infection; and (iii) study molecular mechanisms of host susceptibility to Trypanosoma brucei brucei infection., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Malaria begins when Plasmodium-infected Anopheles mosquitoes take a blood meal on a vertebrate. During the initial probing process, mosquitoes inject saliva and sporozoites into the host skin. Components of mosquito saliva have the potential to influence sporozoite functionality. Sporozoite-associated mosquito saliva protein 1 (SAMSP1; AGAP013726) was among several proteins identified when sporozoites were isolated from saliva, suggesting it may have an effect on Plasmodium. Recombinant SAMSP1 enhanced sporozoite gliding and cell traversal activity in vitro. Moreover, SAMSP1 decreased neutrophil chemotaxis in vivo and in vitro, thereby also exerting an influence on the host environment in which the sporozoites reside. Active or passive immunization of mice with SAMSP1 or SAMSP1 antiserum diminished the initial Plasmodium burden after infection. Passive immunization of mice with SAMSP1 antiserum also added to the protective effect of a circumsporozoite protein monoclonal antibody. SAMSP1 is, therefore, a mosquito saliva protein that can influence sporozoite infectivity in the vertebrate host., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Malaria remains a major cause of mortality in the world and an efficient vaccine is the best chance of reducing the disease burden. Vaccination strategies for the liver stage of disease that utilise injection of live radiation-attenuated sporozoites (RAS) confer sterile immunity, which is mediated by CD8 + memory T cells, with liver-resident memory T cells (T RM ) being particularly important. We have previously described a TCR transgenic mouse, termed PbT-I, where all CD8 + T cells recognize a specific peptide from Plasmodium. PbT-I form liver T RM cells upon RAS injection and are capable of protecting mice against challenge infection. Here, we utilize this transgenic system to examine whether nonliving sporozoites, killed by heat treatment (HKS), could trigger the development of Plasmodium-specific liver T RM cells. We found that HKS vaccination induced the formation of memory CD8 + T cells in the spleen and liver, and importantly, liver T RM cells were fewer in number than that induced by RAS. Crucially, we showed the number of T RM cells was significantly higher when HKS were combined with the glycolipid α-galactosylceramide as an adjuvant. In the future, this work could lead to development of an antimalaria vaccination strategy that does not require live sporozoites, providing greater utility., (© 2021 Wiley-VCH GmbH.)

Transmission of malaria-causing parasites to and by the mosquito relies on active parasite migration and constitutes bottlenecks in the Plasmodium life cycle. Parasite adaption to the biochemically and physically different environments must hence be a key evolutionary driver for transmission efficiency. To probe how subtle but physiologically relevant changes in environmental elasticity impact parasite migration, we introduce 2D and 3D polyacrylamide gels to study ookinetes, the parasite forms emigrating from the mosquito blood meal and sporozoites, the forms transmitted to the vertebrate host. We show that ookinetes adapt their migratory path but not their speed to environmental elasticity and are motile for over 24 h on soft substrates. In contrast, sporozoites evolved more short-lived rapid gliding motility for rapidly crossing the skin. Strikingly, sporozoites are highly sensitive to substrate elasticity possibly to avoid adhesion to soft endothelial cells on their long way to the liver. Hence, the two migratory stages of Plasmodium evolved different strategies to overcome the physical challenges posed by the respective environments and barriers they encounter., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

During five years, from 1953, a village scale indoors residual spraying (IRS) was done in the pilot zone of Bobo-Dioulasso, Burkina Faso, with DDT or dieldrin (DLN) or even HCH with a conceptually both entomological and parasitological evaluation [18].Compared to the control area, DDT induced an approximatively 95% and 67% reduction in the landing rate of Anopheles gambiae , respectively inside and outside human houses but due to its irritant action, DDT greatly increased their exophagic behaviour. However, DLN had no impact on the landing rate of An. gambiae either indoors or outdoors due to the already noticed resistance of this species to this insecticide. The sporozoitic index of An. gambiae was reduced by 96% in the DDT treated areas and by 70% in the DLN treated area.DDT reduced the landing rates of Anopheles funestus by 98% and 91%, inside and outside treated houses respectively. With DLN, these reductions were 98% and 97%, respectively. The sporozoitic index of An. funestus was reduced by 95% in areas treated with DDT.Thus, vector control has reduced malaria transmission due to the two main vectors, An. gambiae and An. funestus , by some 99.8% in DDT treated villages compared to control villages. DLN reduced transmission from An. funestus by 99.9%, but almost not from An. gambiae . Overall, the implementation of vector control based on indoor residual spraying with DDT or DLN reduced by 99.9% the transmission of human Plasmodium in the villages of the pilot zone and therefore the program can be considered as entomologically successful.In children aged 2-9 years (target group for endemicity indices) the splenic index was 84.3% (n = 979) in the control area and 44.4% (n = 8920) in the treated areas (difference -47.3%), the plasmodial prevalence was 60.6% (n = 946) in the control zone and 38.0% (n = 7242) in the treated zones (difference - 37%) but the relatively high level of plasmodic or splenic index in treated villages showed that transmission was maintained at such a level that the program could be considered as a "semi-failure".Besides, the gametocytic indices remained at the same levels (3.28%, n = 946 in the control zone and 3.04%, n = 7242 in the treated zones) indicating the maintenance of the "reservoir of parasites" and the remaining possibilities of transmission.Compared to the control area, the index of new contamination was significantly lower in infants 0-3 months and 4 to 6 months in DDT treated villages but not in infants 7 to 12 months demonstrating that the control vector had some efficacy in the prevention of plasmodial infection but "all newborns were infected within one year" demonstrating that P. falciparum transmission was not completely stopped.In spite of its striking drop, the transmission was not fully stopped, and the programme was considered as a "semi-failure" or even a "failure" and inducing a complete shift in malaria control policy from vector control to mass drug chemotherapy (with several drugs, chloroquine, primaquine, pyriméthamine etc) without complete stop of transmission either. In fact, such vector control operations by DDT may have different analysis; in one side they can be considered an entomological success but, in another side, the actual reduction of plasmodic and splenic indices was not enough to be considered as successful. It was clear that both vector and parasite must be implemented in an integrated programme taking care of insecticide and drug resistance. Nevertheless, such programme, even not as successful as expected, could be considered as encouraging and not "disappointing" as it was. Important lessons can be learned from such large-scale field trial in spite of several methodological and operational issues., (Copyright © 2021 SFMTSI.)

Malaria remains a devastating global health problem, resulting in many annual deaths due to the complications of severe malaria. However, in endemic regions, individuals can acquire 'clinical immunity' to malaria, characterized by a decrease in severe malaria episodes and an increase of asymptomatic Plasmodium falciparum infections. Recently, it has been reported that tolerance to 'clinical malaria' and reduced disease severity correlates with a decrease in the numbers of circulating Vγ9Vδ2 T cells, the major subset of γδ T cells in the human peripheral blood. This is particularly interesting as this population typically undergoes dramatic expansions during acute Plasmodium infections and was previously shown to play antiparasitic functions. Thus, regulated γδ T-cell responses may be critical to balance immune protection with severe pathology, particularly as both seem to rely on the same pro-inflammatory cytokines, most notably TNF and IFN-γ. This has been clearly demonstrated in mouse models of experimental cerebral malaria (ECM) based on Plasmodium berghei ANKA infection. Furthermore, our recent studies suggest that the natural course of Plasmodium infection, mimicked in mice through mosquito bite or sporozoite inoculation, includes a major pathogenic component in ECM that depends on γδ T cells and IFN-γ production in the asymptomatic liver stage, where parasite virulence is seemingly set and determines pathology in the subsequent blood stage. Here, we discuss these and other recent advances in our understanding of the complex-protective versus pathogenic-functions of γδ T cells in malaria., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Plasmodium sporozoites are transmitted to mammals by anopheline mosquitoes and first infect the liver, where they transform into replicative exoerythrocytic forms, which subsequently release thousands of merozoites that invade erythrocytes and initiate the malaria disease. In some species, sporozoites can transform into dormant hypnozoites in the liver, which cause malaria relapses upon reactivation. Transmission from the insect vector to a mammalian host is a critical step of the parasite life cycle, and requires tightly regulated gene expression. Sporozoites are formed inside oocysts in the mosquito midgut and become fully infectious after colonization of the insect salivary glands, where they remain quiescent until transmission. Parasite maturation into infectious sporozoites is associated with reprogramming of the sporozoite transcriptome and proteome, which depends on multiple layers of transcriptional and post-transcriptional regulatory mechanisms. An emerging scheme is that gene expression in Plasmodium sporozoites is controlled by alternating waves of transcription activity and translational repression, which shape the parasite RNA and protein repertoires for successful transition from the mosquito vector to the mammalian host., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Briquet, Marinach, Silvie and Vaquero.)

Investigations of malaria infection are often conducted by studying rodent Plasmodium species in inbred laboratory mice, but the efficacy of vaccines or adjunctive therapies observed in these models often does not translate to protection in humans. This raises concerns that mouse malaria models do not recapitulate important features of human malaria infections. African woodland thicket rats ( Grammomys surdaster ) are the natural host for the rodent malaria parasite Plasmodium berghei and the suspected natural host for Plasmodium vinckei vinckei . Previously, we reported that thicket rats are highly susceptible to diverse rodent parasite species, including P. berghei , Plasmodium yoelii , and Plasmodium chabaudi chabaudi , and are a more stringent model to assess the efficacy of whole-sporozoite vaccines than laboratory mice. Here, we compare the course of infection and virulence with additional rodent Plasmodium species, including various strains of P. berghei , P. yoelii , P. chabaudi , and P. vinckei , in thicket rats versus laboratory mice. We present evidence that rodent malaria parasite growth typically differs between the natural versus nonnatural host; G. surdaster limit infection by multiple rodent malaria strains, delaying and reducing peak parasitemia compared with laboratory mice. The course of malaria infection in thicket rats varied depending on parasite species and strain, resulting in self-cure, chronic parasitemia, or rapidly lethal infection, thus offering a variety of rodent malaria models to study different clinical outcomes in the natural host.

Complex experimental studies of vertebrate host, vector, and parasite interactions are essential in understanding virulence, but are difficult or impossible to conduct if vector species are unknown. Subinoculation of erythrocytic meronts of avian malarial parasites into susceptible hosts can avoid this problem, but this approach omits early exoerythrocytic stages, e.g. cryptozoites and metacryptozoites, that normally develop from sporozoites. A fundamental question that has remained unanswered is whether blood stage and sporozoite-induced malarial infections lead to differences in the dynamics of parasitemia in acute infections, patterns of parasite development, and host mortality. Here we demonstrate in a Carduelis spinus - Plasmodium relictum (genetic lineage pSGS1) system that experimental infections using inoculation of infected blood and using mosquito bite show similar peaks of parasitemias, but some measures of parasite development in the vertebrate host differ. Infected birds from all groups show decreased activity during the peak of parasitemia. There is no doubt that experimental infections using vectors provide the most precise information about the development of a parasite and its virulence in the host, but experimental infections using blood stages demonstrate similar parasitemias and effects on the host. These results are important for further experimental research of malarial parasites, especially studying avian Plasmodium parasites with unknown vectors., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The physicochemical properties of an antigen (Ag) influence the type, specificity, as well as duration of emerging immune responses. Like immune responses arising to nominal protein Ags, reactivities to protozoan parasites, Plasmodium falciparum and P. berghei, the causative agents of human and mouse malaria, respectively, are shaped by the form of the parasite. While repeated natural exposures to infectious Plasmodium sporozoites (spzs) typically induce malaria, immunizations with radiation or genetically attenuated forms of Plasmodium spzs induce sterile and durable protective immunity. The immune mechanisms that are responsible for these diametrically opposite outcomes are still not well understood. It has been observed that infectious spzs engage in mechanisms that evade immune recognition and thus prevent protective immune responses from occurring. The responses that develop are characteristic of anti-disease immunity; acquisition of protective immunity against infection is a prolonged process, and it decays once exposure to the parasite ceases. In contrast, repeated exposures to attenuated Plasmodium spzs induce antibodies and CD4 T cells directed primarily to the spz surface Ags and effector and memory CD8 T cells that localize in the liver and are specific for Plasmodium liver-stage Ags. Understanding the precise mechanisms, including early interactions between the spzs and Ag-presenting cells that lead to the manner of Ag processing and presentation, are of key importance as such information would substantially contribute to the successful development of malaria vaccine.

The ability to monitor micropipette injections with a high-resolution fluorescent microscope has utility for a variety of applications. Herein, different approaches were tested for creating broad-band fluorescently labelled glass micropipettes including: UV cured glass glues, baked glass enamel containing fluorescent dyes as well as nanodiamonds attached during pipette formation in the microforge. The most robust and simplest approach was to use labelled baked enamel on the exterior of the pipette. This approach was tested using pipettes designed to mimic a mosquito proboscis for the injection of the malaria parasite, Plasmodium spp., into the dermis of a living mouse ear. The pipette (∼30 micron diameter) was easily detected in the microscopy field of view and tolerated multiple insertions through the skin. This simple inexpensive approach to fluorescently labelling micropipettes will aid in the development of procedures under the fluorescent microscope., (© 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.)

The efficiency of malaria parasite development within mosquito vectors (sporogony) is a critical determinant of transmission. Sporogony is thought to be controlled by environmental conditions and mosquito/parasite genetic factors, with minimal contribution from mosquito behaviour during the period of parasite development. We tested this assumption by investigating whether successful sporogony of Plasmodium falciparum parasites through to human-infectious transmission stages is influenced by the host species upon which infected mosquitoes feed. Studies were conducted on two major African vector species that generally are found to differ in their innate host preferences: Anopheles arabiensis and An. gambiae sensu stricto. We show that the proportion of vectors developing transmissible infections (sporozoites) was influenced by the source of host blood consumed during sporogony. The direction of this effect was associated with the innate host preference of vectors: higher sporozoite prevalences were generated in the usually human-specialist An. gambiae s.s. feeding on human compared to cow blood, whereas the more zoophilic An. arabiensis had significantly higher prevalences after feeding on cow blood. The potential epidemiological implications of these results are discussed.

Background: The Thai-Myanmar border is a remaining hotspot for malaria transmission. Malaria transmission in this region continues year-round, with a major peak season in July-August, and a minor peak in October-November. Malaria elimination requires better knowledge of the mosquito community structure, dynamics and vectorial status to support effective vector control., Methods: Adult Anopheles mosquitoes were collected using CDC light traps and cow bait in 7 villages along the Thai-Myanmar border in January 2011 - March 2013. Mosquitoes were determined to species by morphological characters. Plasmodium-positivity was determined by circumsporozoite protein ELISA., Results: The 2986 Anopheles mosquitoes collected were assigned to 26 species, with Anopheles minimus sensu lato (s.l.) (40.32%), An. maculatus s.l. (21.43%), An. annularis s.l. (14.43%), An. kochi (5.39%), An. tessellatus (5.26%), and An. barbirostris s.l. (3.52%) being the top six most abundant species. Plasmodium-infected mosquitoes were found in 22 positive samples from 2906 pooled samples of abdomens and heads/thoraxes. Four mosquito species were found infected with Plasmodium: An. minimus s.l., An. maculatus s.l., An. annularis s.l. and An. barbirostris s.l. The infectivity rates of these mosquitoes were 0.76, 0.37, 0.72, and 1.74%, respectively. Consistent with a change in malaria epidemiology to the predominance of P. vivax in this area, 20 of the 22 infected mosquito samples were P. vivax-positive. The four potential vector species all displayed apparent seasonality in relative abundance. While An. minimus s.l. was collected through the entire year, its abundance peaked in the season immediately after the wet season. In comparison, An. maculatus s.l. numbers showed a major peak during the wet season. The two potential vector species, An. annularis s.l. and An. barbirostris s.l., both showed peak abundance during the transition from wet to dry season. Moreover, An. minimus s.l. was more abundant in indoor collections, whereas An. annularis s.l. and An. barbirostris s.l. were more abundant in outdoor collections, suggesting their potential role in outdoor malaria transmission., Conclusions: This survey confirmed the major vector status of An. minimus s.l. and An. maculatus s.l. and identified An. annularis s.l. and An. barbirostris s.l. as additional vectors with potential importance in malaria transmission after the wet season.

TLRs (Toll-like receptors) play an important role in the initiation of innate immune responses against invading microorganisms. Although several TLRs have been reported to be involved in the innate immune response against the blood-stage of malaria parasites, the role of TLRs in the development of the pre-erythrocytic stage is still largely unknown. Here, we found that sporozoite and its lysate could significantly activate the TLR2, and induce macrophages to release proinflammatory cytokines, including IL-6, MCP-1 and TNF-α, in a TLR2-dependent manner. Further studies showed that sporozoite and its lysate could be recognized by either TLR2 homodimers or TLR2/1 and TLR2/6 heterodimers, implicating the complexity of TLR2 agonist in sporozoite. Interestingly, the TLR2 signaling can significantly suppress the development of the pre-erythrocytic stage of Plasmodium yoelii, as both liver parasite load and subsequent parasitemia were significantly elevated in both TLR2- and MyD88-deficient mice. Additionally, the observed higher level of parasite burden in TLR2(-/-) mice was found to be closely associated with a reduction in proinflammatory cytokines in the liver. Therefore, we provide the first evidence that sporozoites can activate the TLR2 signaling, which in turn significantly inhibits the intrahepatic parasites. This may provide us with novel clues to design preventive anti-malaria therapies.

Plasmodium relictum (lineage pGRW4) causes malaria in birds and is actively transmitted in countries with warm climates and also temperate regions of the New World. In Europe, the lineage pGRW4 has been frequently reported in many species of Afrotropical migrants after their arrival from wintering grounds, but is rare in European resident birds. Obstacles for transmission of this parasite in Europe have not been identified. Culex quinquefasciatus is an effective vector of pGRW4 malaria, but this mosquito is absent from temperate regions of Eurasia. It remains unclear if the lineage pGRW4 completes sporogony in European species of mosquitoes. Here we compare the sporogonic development of P. relictum (pGRW4) in experimentally infected mosquitoes Culex pipiens pipiens form molestus, C. quinquefasciatus, and Ochlerotatus cantans. The pGRW4 parasite was isolated from a garden warbler Sylvia borin, multiplied, and used to infect laboratory-reared Culex spp. and wild-caught Ochlerotatus mosquitoes by allowing them to take blood meals on infected birds. The exposed females were maintained at a mean laboratory temperature of 19 °C, which ranged between 14 °C at night and 24 °C during daytime. They were dissected on intervals to study the development of sporogonic stages. Only ookinetes developed in O. cantans; sporogonic development was abortive. The parasite completed sporogony in both Culex species, with similar patterns of development, and sporozoites were reported in the salivary glands 16 days after infection. The presence of sporogonic stages of the lineage pGRW4 in mosquitoes was confirmed by PCR-based testing of (1) the sporozoites present in salivary glands and (2) the single oocysts, which were obtained by laser microdissection from infected mosquito midguts. This study shows that P. relictum (pGRW4) completes sporogony in C. p. pipiens at relatively low temperatures. We conclude that there are no restrictions for spreading this bird infection in Europe from the point of view of vector availability and temperature necessary for sporogony. Other factors should be considered and were discussed for the explanation of rare reports of this malaria parasite in Europe.

Current views about the impact of Wolbachia on Plasmodium infections are almost entirely based on data regarding artificially transfected mosquitoes. This work has shown that Wolbachia reduces the intensity of Plasmodium infections in mosquitoes, raising the exciting possibility of using Wolbachia to control or limit the spread of malaria. Whether natural Wolbachia infections have the same parasite-inhibiting properties is not yet clear. Wolbachia-mosquito combinations with a long evolutionary history are, however, key for understanding what may happen with Wolbachia-transfected mosquitoes after several generations of coevolution. We investigate this issue using an entirely natural mosquito-Wolbachia-Plasmodium combination. In contrast to most previous studies, which have been centred on the quantification of the midgut stages of Plasmodium, we obtain a measurement of parasitaemia that relates directly to transmission by following infections to the salivary gland stages. We show that Wolbachia increases the susceptibility of Culex pipiens mosquitoes to Plasmodium relictum, significantly increasing the prevalence of salivary gland stage infections. This effect is independent of the density of Wolbachia in the mosquito. These results suggest that naturally Wolbachia-infected mosquitoes may, in fact, be better vectors of malaria than Wolbachia-free ones.

Objective: To identify the biological forms, sporozoite rate and molecular characterization of the Anopheles stephensi (An. stephensi) in Hormozgan and Sistan-Baluchistan provinces, the most important malarious areas in Iran., Methods: Wild live An. stephensi samples were collected from different malarious areas in southern Iran. The biological forms were identified based on number of egg-ridges. Molecular characterization of biological forms was verified by analysis of the mitochondrial cytochrome oxidase subunit I and II (mtDNA-COI/COII). The Plasmodium infection was examined in the wild female specimens by species-specific nested-PCR method., Results: Results showed that all three biological forms including mysorensis, intermediate and type are present in the study areas. Molecular investigations revealed no genetic variation between mtDNA COI/COII sequences of the biological forms and no Plasmodium parasites was detected in the collected mosquito samples., Conclusions: Presence of three biological forms with identical sequences showed that the known biological forms belong to a single taxon and the various vectorial capacities reported for these forms are more likely corresponded to other epidemiological factors than to the morphotype of the populations. Lack of malaria parasite infection in An. stephensi, the most important vector of malaria, may be partly due to the success and achievement of ongoing active malaria control program in the region., (Copyright © 2014 Asian Pacific Tropical Biomedical Magazine. Published by Elsevier B.V. All rights reserved.)

Background & Objectives: A new irrigation canal system is under construction in Dhenkanal district of Odisha, to increase the production of rice crop and thereby improve the living standard of farmers in the project area. Construction of canal may increase the transmission of malaria by creating vector breeding habitats. Knowledge about bionomics of vectors will support authorities for appropriate management of the disease in a changing ecological set up. The aim of this study was to assess the malaria transmission in the bank of the canal area under construction., Methods: The entomological survey was carried out in three seasons, winter, summer and rainy during the period November 2008-October 2010 in the study area. Adult mosquitoes were collected by using suction tubes and flash lights. Mosquito species identification was done by using standard keys, separated according to abdominal conditions and were kept in an isopropanol for further molecular analysis of sibling species, presence of sporozoites and human blood meal. Larvae were collected by dippers and reared in the laboratory, and the emerged adults were identified to species. The epidemiology of malaria was evaluated from the data collected by the State Health Department. Insecticide succeptibility test was done by WHO method., Results: The adult mosquito collection from the study area showed the prevalence of 14 species belonging to three genera, i.e. Anopheles, Culex and Aedes. The per man hour densities (PMHD) of An. culicifacies were 3.8, 1.4, 4.8; that of An. annularis were 2.1, 1, 2.1; and that of An. fluviatilis were 1.4, 0.3, 0.6 during winter, summer and rainy seasons respectively. Sibling species identified were: An. culicifacies A, B, C and D, An. annularis A and An. fluviatilis S. Sporozoite rates of An. culicifacies A and C were 1.1 and 0.5% respectively and that of An. annularis A was 2% (reported for the first time in the state). Both the vectors (An. culicifacies and An. annularis) showed resistance to DDT and malathion and were susceptible to deltamethrin, whereas An. fluviatilis was susceptible to all the three insecticides tested., Interpretation & Conclusion: Anopheles culicifacies, An. fluviatilis and An. annularis were prevalent in all the three seasons. The artificial ponds and seepage pools of canal are the major breeding sites for An. culicifacies and An. annularis. Thus, in the canal command area, control of malaria transmission requires use of insecticide-treated bednets and use of biolarvicides (seepage pools) and larvivorous fish (artificial ponds) wherever feasible.