Parasite Plasmodium Research Articles

Novel hydrazone compounds with broad-spectrum antiplasmodial activity and synergistic interactions with antimalarial drugs.

The development of novel antiplasmodial compounds with broad-spectrum activity against different stages of Plasmodium parasites is crucial to prevent malaria disease and parasite transmission. This study evaluated the antiplasmodial activity of seven novel hydrazone compounds (referred to as CB compounds: CB-27, CB-41, CB-50, CB-53, CB-58, CB-59, and CB-61) against multiple stages of Plasmodium parasites. All CB compounds inhibited blood stage proliferation of drug-resistant or sensitive strains of Plasmodium falciparum in the low micromolar to nanomolar range. Interestingly, CB-41 exhibited prophylactic activity against hypnozoites and liver schizonts in Plasmodium cynomolgi, a primate model for Plasmodium vivax. Four CB compounds (CB-27, CB-41, CB-53, and CB-61) inhibited P. falciparum oocyst formation in mosquitoes, and five CB compounds (CB-27, CB-41, CB-53, CB-58, and CB-61) hindered the in vitro development of Plasmodium berghei ookinetes. The CB compounds did not inhibit the activation of P. berghei female and male gametocytes in vitro. Isobologram assays demonstrated synergistic interactions between CB-61 and the FDA-approved antimalarial drugs, clindamycin and halofantrine. Testing of six CB compounds showed no inhibition of Plasmodium glutathione S-transferase as a putative target and no cytotoxicity in HepG2 liver cells. CB compounds are promising candidates for further development as antimalarial drugs against multidrug-resistant parasites, which could also prevent malaria transmission.

Cyclic peroxides and analogs: Antibacterial, antimalarial, and cytotoxic marine products from Xisha sponge Diacarnus sp.

A chemical investigation of the dichloromethane extract from the Xisha sponge Diacarnus sp. revealed seven undescribed norterpene cyclic peroxides, named diacarperoxides T–Z, and five unreported related norterpenes, named diacarnoids E–I, and eleven previously reported compounds. The structures of these isolated compounds, including their absolute configurations, were elucidated based on extensive spectroscopic analyses, electronic circular dichroism (ECD) calculations, Snatzke's method, [Rh2(OCOCF3)4]-induced ECD spectra, and modified Mosher's method. Bioassays were performed to assess the antibacterial activity against six pathogenic bacteria, cytotoxicities toward three cancer cell lines, and antimalarial activity against Plasmodium parasites. Most of the cyclic peroxides exhibited substantial antibacterial activity (MIC 1–8 μg/mL). Diacarperoxide W and nuapapuin A showed substantial antimalarial activity with IC50 values of 0.98 and 2.83 μM. Moreover, many compounds exhibited <50% cell survival rates, and IC50 values of 0.22–6.33 μM. The apoptosis assay showed that nuapapuin A induced cancer cell apoptosis in a dose-dependent manner.

Machine learning for predicting Plasmodium liver stage development in vitro using microscopy imaging

Malaria, a significant global health challenge, is caused by Plasmodium parasites. The Plasmodium liver stage plays a pivotal role in the establishment of the infection. This study focuses on the liver stage development of the model organism Plasmodium berghei, employing fluorescent microscopy imaging and convolutional neural networks (CNNs) for analysis. Convolutional neural networks have been recently proposed as a viable option for tasks such as malaria detection, prediction of host-pathogen interactions, or drug discovery. Our research aimed to predict the transition of Plasmodium-infected liver cells to the merozoite stage, a key development phase, 15 hours in advance. We collected and analyzed hourly imaging data over a span of at least 38 hours from 400 sequences, encompassing 502 parasites. Our method was compared to human annotations to validate its efficacy. Performance metrics, including the area under the receiver operating characteristic curve (AUC), sensitivity, and specificity, were evaluated on an independent test dataset. The outcomes revealed an AUC of 0.873, a sensitivity of 84.6%, and a specificity of 83.3%, underscoring the potential of our CNN-based framework to predict liver stage development of P. berghei. These findings not only demonstrate the feasibility of our methodology but also could potentially contribute to the broader understanding of parasite biology.

Identification of novel peptide inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR): molecular docking and MD simulation studies

The presence of drug-resistant variants of Plasmodium parasites within the population has presented a substantial obstacle to the eradication of Malaria. As a result, numerous research groups have directed their efforts towards creating new medication candidates that specifically target parasites. In this study, our main objective was to identify tri-peptide inhibitors for Plasmodium falciparum Dihydrofolate Reductase (PfDHFR) with the aim of finding a new peptide that exhibits superior binding properties compared to the current inhibitor, WR99210. In order to achieve this objective, a virtual library consisting of 8000 tripeptides was generated and subjected to computational screening against wild-type PfDHFR. The purpose of this screening was to discover the most effective binders at the active site. The four most optimal tripeptides identified (Trp-Trp-Glu, Trp-Phe-Tyr, Phe-Trp-Trp, Tyr-Trp-Trp) exhibited significant non-covalent interactions inside the active site of PfDHFR and had binding energies ranging from −9.5 to −9.0 kcal/mol and WR99210 had a binding energy of −6.2 kcal/mol. A 250 ns Molecular Dynamics (MD) simulation was performed to investigate the kinetic and thermodynamic characteristics of the protein-ligand complexes. The Root Mean Square Deviation (RMSD) values for the optimal tripeptides fell within the allowed range, indicating the stability of the ligands inside the protein complex. The Ki value for the most effective tripeptide was 0.3482 µM, whereas WR99210 had a Ki value of 1.02 µM. This article presents the initial discovery of peptide inhibitors targeting PfDHFR. In this text, we provide a comprehensive explanation of the interactions that occur between peptides and the enzyme. Communicated by Ramaswamy H. Sarma

Plasmodium yoelii surface-related antigen (PySRA) modulates the host pro-inflammatory responses via binding to CD68 on macrophage membrane.

Malaria, one of the major infectious diseases in the world, is caused by the Plasmodium parasite. Plasmodium antigens could modulate the inflammatory response by binding to macrophage membrane receptors. As an export protein on the infected erythrocyte membrane, Plasmodium surface-related antigen (SRA) participates in the erythrocyte invasion and regulates the immune response of the host. This study found that the F2 segment of P. yoelii SRA activated downstream MAPK and NF-κB signaling pathways by binding to CD68 on the surface of the macrophage membrane and regulating the inflammatory response. The anti-PySRA-F2 antibody can protect mice against P. yoelii, and the pro-inflammatory responses such as IL-1β, TNF-α, and IL-6 after infection with P. yoelii are attenuated. These findings will be helpful for understanding the involvement of the pathogenic mechanism of malaria with the exported protein SRA.

The Plasmodium vivax MSP1P-19 is involved in binding of reticulocytes through interactions with the membrane proteins band3 and CD71

The parasite Plasmodium vivax preferentially invades human reticulocytes. Its merozoite surface protein 1 paralog (PvMSP1P), particularly the 19-kDa C-terminal region (PvMSP1P-19), has been shown to bind to reticulocytes and this binding can be inhibited by antisera obtained by PvMSP1P-19 immunization. The molecular mechanism of interactions between PvMSP1P-19 and reticulocytes during P. vivax invasion, however, remains unclear. In this study, we analyzed the ability of MSP1P-19 to bind to different concentrations of reticulocytes and confirmed its reticulocyte preference. LC-MS analysis was used to identify two potential reticulocyte receptors, band3 and CD71, that interact with MSP1P-19. Both PvMSP1P-19 and its sister taxon Plasmodium cynomolgi MSP1P-19 (PcMSP1P-19) were found to bind to the extracellular loop (loop 5) of band3, where the interaction of MSP1P-19 with band3 was chymotrypsin-sensitive. Antibodies against band3-P5, CD71, and MSP1P-19 reduced the binding activity of PvMSP1P-19 and PcMSP1P-19 to reticulocytes, while MSP1P-19 proteins inhibited P. falciparum invasion in vitro in a concentration-dependent manner. To sum up, identification and characterization of the reticulocyte receptor is important for understanding the binding of reticulocytes by MSP1P-19.

Study of Mosquito Borne Deasease Found in Yavatmal Region

Abstract: Mosquito-borne diseases are a significant public health concern in the Yavatmal region of Maharashtra, India. Malaria, dengue fever, and Japanese encephalitis are among the prominent diseases transmitted by mosquitoes in this region. Malaria, caused by the Plasmodium parasite, is endemic in Yavatmal, with transmission occurring mainly during the monsoon and post-monsoon seasons. Dengue fever, caused by the dengue virus and transmitted by Aedes mosquitoes, has seen periodic outbreaks in the region, with increased incidence during the rainy season. Japanese encephalitis, caused by the Japanese encephalitis virus and transmitted by Culex mosquitoes, poses a threat to the population, particularly in rural areas. Several factors contribute to the spread of mosquito-borne diseases in the Yavatmal region, including inadequate sanitation, stagnant water bodies, deforestation, and climate change. Socioeconomic factors such as poverty and limited access to healthcare further exacerbate the problem. Control and prevention efforts in the region primarily focus on vector control measures, including insecticide spraying, larval source management, and the use of bed nets. Community education and awareness campaigns play a crucial role in promoting preventive behaviors such as the use of insect repellents and proper waste disposal. Challenges in addressing mosquito-borne diseases in Yavatmal include inadequate healthcare infrastructure, insufficient funding, and the emergence of insecticide resistance among mosquito populations. Collaborative efforts involving government agencies, healthcare providers, researchers, and community organizations are essential to effectively combat these diseases. Strengthening surveillance systems, improving access to diagnostic and treatment facilities, and implementing integrated vector management strategies are necessary steps towards reducing the burden of mosquito-borne diseases in the Yavatmal region and improving public health outcomes.

Malaria parasitic detection using a new Deep Boosted and Ensemble Learning framework

Malaria is a potentially fatal plasmodium parasite injected by female anopheles mosquitoes that infect red blood cells and cause millions of lifelong disability worldwide yearly. However, specialists’ manual screening in clinical practice is laborious and prone to error. Therefore, a novel Deep Boosted and Ensemble Learning (DBEL) framework, comprising the stacking of new Boosted-BR-STM convolutional neural networks (CNN) and the ensemble ML classifiers, is developed to screen malaria parasite images. The proposed Boosted-BR-STM is based on a new dilated-convolutional block-based Split Transform Merge (STM) and feature-map Squeezing–Boosting (SB) ideas. Moreover, the new STM block uses regional and boundary operations to learn the malaria parasite’s homogeneity, heterogeneity, and boundary with patterns. Furthermore, the diverse boosted channels are attained by employing Transfer Learning-based new feature-map SB in STM blocks at the abstract, medium, and conclusion levels to learn minute intensity and texture variation of the parasitic pattern. Additionally, to enhance the learning capacity of Boosted-BR-STM and foster a more diverse representation of features, boosting at the final stage is achieved through TL by utilizing multipath residual learning. The proposed DBEL framework implicates the stacking of prominent and diverse boosted channels and provides the generated discriminative features of the developed Boosted-BR-STM to the ensemble of ML classifiers. The proposed framework improves the discrimination ability and generalization of ensemble learning. Moreover, the deep feature spaces of the developed Boosted-BR-STM and customized CNNs are fed into ML classifiers for comparative analysis. The proposed DBEL framework outperforms the existing techniques on the NIH malaria dataset that are enhanced using discrete wavelet transform to enrich feature space. The proposed DBEL framework achieved Accuracy (98.50%), Sensitivity (0.9920), F-score (0.9850), and AUC (0.9960), which suggests it to be utilized for malaria parasite screening.

Association of Hematological Parameters with Malaria Positive Patients in District Bannu

Background: Malaria is one of the most widespread parasitic blood infections caused by plasmodium parasite. Well known feature of malaria is change in hematological parameters such as red blood cell, hemoglobin, platelets, white blood cell and differential count are significantly low. This study aim was to find the association of malaria with Complete Blood Count in malaria positive patient. Methods: A six-month cross-sectional research was carried out in the Bannu district. Venous blood samples from 500 individuals were taken and placed in EDTA tubes for smear preparation and total blood counts. The Sysmex Hematology Analyzer was used to evaluate blood counts. Using SPSS-22, all data were calculated. Results: Among total, 60 Malaria positive patient samples were processed for association of malaria with CBC. Out of total, male were 78% and female were 22%. Malaria positive patient with anaemia were found 53% in which male and female were 81% and 18% respectively. Thrombocytopenia were found in 16% in which male were 60% and 40% female patients. Malaria positive with abnormal TLC were observed in 11% patients (male 8%, female 3%). Moreover, Malaria positive with abnormal DLC were found 70% (male 73%, female 26%). Conclusion: This study shows a significant change in hematological parameter in Malaria patients. Thrombocytopenia is second most common hematological abnormality. In TLC abnormality, rare cases of leukocytosis were noted. In malaria-endemic regions, hematological indicators may be a suitable and reliable adjuvant in the early identification of individuals with mild-to-severe parasitemia.

LC3B labeling of the parasitophorous vacuole membrane of Plasmodium berghei liver stage parasites depends on the V-ATPase and ATG16L1.

The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra-hepatic development before initiating a blood-stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterized by a long-lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V-ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites.

PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum.

Mitosis is a fundamental process for Plasmodium parasites, which plays a vital role in their survival within two distinct hosts-human and Anopheles mosquitoes. Despite its great significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, mitosis is regulated by one of the pivotal complexes known as condensin complexes. The condensin complexes are responsible for chromosome condensation, ensuring the faithful distribution of genetic material to daughter cells. While condensin complexes have recently been identified in Plasmodium spp., our understanding of how this complex is assembled and its precise functions during the blood stage development of Plasmodium falciparum remains largely unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood stage development of P. falciparum. Our findings reveal that PfCAP-H is essential and plays a pivotal role in upholding the structure of condensin I and facilitating karyokinesis.

Role of TAM Receptors in Antimalarial Humoral Immune Response.

Immune response against malaria and the clearance of Plasmodium parasite relies on germinal-center-derived B cell responses that are temporally and histologically layered. Despite a well-orchestrated germinal center response, anti-Plasmodium immune response seldom offers sterilizing immunity. Recent studies report that certain pathophysiological features of malaria such as extensive hemolysis, hypoxia as well as the extrafollicular accumulation of short-lived plasmablasts may contribute to this suboptimal immune response. In this review, we summarize some of those studies and attempt to connect certain host intrinsic features in response to the malarial disease and the resultant gaps in the immune response.

Global profiling of protein S-palmitoylation in the second-generation merozoites of Eimeria tenella.

The intracellular protozoan Eimeria tenella is responsible for avian coccidiosis which is characterized by host intestinal damage. During developmental cycle, E. tenella undergoes versatile transitional stages such as oocyst, sporozoites, merozoites, and gametocytes. These developmental transitions involve changes in cell shape and cell size requiring cytoskeletal remodeling and changes in membrane proteins, which may require transcriptional and translational regulations as well as post-translational modification of proteins. Palmitoylation is a post-translational modification (PTM) of protein that orchestrates protein targeting, folding, stability, regulated enzymatic activity and even epigenetic regulation of gene expression. Previous research revealed that protein palmitoylation play essential role in Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, and several Plasmodium parasites. Until now, there is little information on the enzymes related to palmitoylation and role of protein acylation or palmitoylation in E. tenella. Therefore, palmitome of the second-generation merozoite of E. tenella was investigated. We identified a total of 2569 palmitoyl-sites that were assigned to 2145 palmitoyl-peptides belonging to 1561 protein-groups that participated in biological processes including parasite morphology, motility and host cell invasion. In addition, RNA biosynthesis, protein biosynthesis, folding, proteasome-ubiquitin degradation, and enzymes involved in PTMs, carbohydrate metabolism, glycan biosynthesis, and mitochondrial respiratory chain as well as vesicle trafficking were identified. The study allowed us to decipher the broad influence of palmitoylation in E. tenella biology, and its potential roles in the pathobiology of E. tenella infection. Raw data are publicly available at iProX with the dataset identifier PXD045061.

Identification of a novel protein localized to the crystalloid of the Plasmodium ookinete

Reducing Plasmodium parasite transmission via the mosquito vector is a promising strategy for malaria control and elimination in endemic regions. In the mosquito midgut after the ingestion of an infected blood meal, malaria parasite gametes egress from erythrocytes and fertilize to develop into motile ookinetes that traverse midgut epithelial cells and transform into oocysts adjacent the basal lamina. Plasmodium ookinetes and young oocysts possess a unique organelle called the crystalloid; which has a honeycomb-like matrix structure and is indicated to be involved in sporozoite formation and maturation. In this study, we identified a novel crystalloid protein, PY17X_1113800, that is exclusively expressed in developing ookinetes. The protein possesses a signal peptide sequence, but lacks a transmembrane domain or GPI anchor signal sequence, as well as predicted adhesive domains which are characterisitic of many crystalloid proteins. The protein is highly conserved across the phylum Apicomplexa and within the greater clade Alveolata, such as Vitrella and the ciliates Paramecium and Tetrahymena, but is absent in cryptosporidia.

PbARID-associated chromatin remodeling events are essential for gametocyte development in Plasmodium.

Gametocyte development of the Plasmodium parasite is a key step for transmission of the parasite. Male and female gametocytes are produced from a subpopulation of asexual blood-stage parasites, but the mechanisms that regulate the differentiation of sexual stages are still under investigation. In this study, we investigated the role of PbARID, a putative subunit of a SWI/SNF chromatin remodeling complex, in transcriptional regulation during the gametocyte development of P. berghei. PbARID expression starts in early gametocytes before the manifestation of male and female-specific features, and disruption of its gene results in the complete loss of gametocytes with detectable male features and the production of abnormal female gametocytes. ChIP-seq analysis of PbARID showed that it forms a complex with gSNF2, an ATPase subunit of the SWI/SNF chromatin remodeling complex, associating with the male cis-regulatory element, TGTCT. Further ChIP-seq of PbARID in gsnf2-knockout parasites revealed an association of PbARID with another cis-regulatory element, TGCACA. RIME and DNA-binding assays suggested that HDP1 is the transcription factor that recruits PbARID to the TGCACA motif. Our results indicated that PbARID could function in two chromatin remodeling events and paly essential roles in both male and female gametocyte development.

Comparative assessment of malaria diagnostic techniques updates

Malaria is a life-threatening disease caused by Plasmodium parasites that enter the bloodstream via insect vectors, such as mosquitos. The disease has devastated parts of Africa and the wider human population throughout human periods and thus remains a significant global health challenge due to the difficulty in the procurement of a vaccine and the nature of its spread. While many have turned towards preventative methods, the first thing that must happen is a timely and accurate diagnosis of malaria. This is crucial for the effective management and control of the disease. However, the current diagnostic methods have various limitations, including cost, availability, and accuracy. This review aims to unmask the challenges of malaria diagnosis by exploring existing diagnostic techniques and shedding light on emerging technologies that show promise in improving malaria detection. Furthermore, it examines the potential of innovative approaches, such as artificial intelligence (AI) and molecular diagnostics, in transforming malaria diagnosis and contributing to global efforts toward malaria elimination.

A new mechanism of antibody diversity: formation of the natural antibodies containing LAIR1 and LILRB1 extracellular domains.

The recent discovery of public antibodies targeting Plasmodium falciparum-encoded repetitive interspersed families of polypeptides (RIFINs), which contain extracellular immunoglobulin-like domains from LAIR1 or LILRB1, constitutes a significant step forward in comprehending the reactivity of the Plasmodium parasite. These antibodies arise from unique B cell clones and demonstrate extensive cross-reactivity through their interaction with P. falciparum RIFINs. LAIR1 and LILRBs are specialized type I transmembrane glycoproteins, classified as immune inhibitory receptors, restricted to primates and mainly found on hematopoietic cells. They are instrumental in modulating interactions within the tumor microenvironment and across the immune system, and are increasingly recognized as important in anti-cancer immunotherapy and pathogen defense. The presence of LAIR1/LILRB1-containing antibodies offers new insights into malaria parasite evasion strategies and the immune system's response. Additionally, the innovative method of integrating extra exons into the antibody switch region is a noteworthy advancement, enriching the strategies for the generation of a varied array of bispecific and multispecific antibodies.

Convolutional Neural Networks in Malaria Diagnosis: A Study on Cell Image Classification

INTRODUCTION: Malaria, a persistent global health threat caused by Plasmodium parasites, necessitates rapid and accurate identification for effective treatment and containment. This study investigates the utilization of convolutional neural networks (CNNs) to enhance the precision and speed of malaria detection through the classification of cell images infected with malaria.&#x0D; OBJECTIVES: The primary objective of this research is to explore the effectiveness of CNNs in accurately classifying malaria-infected cell images. By employing various deep learning models, including ResNet50, AlexNet, Inception V3, VGG19, VGG16, and MobileNetV2, the study aims to assess the performance of each model and identify their strengths and weaknesses in malaria diagnosis.&#x0D; METHODS: A balanced dataset comprising approximately 8,000 enhanced images of blood cells, evenly distributed between infected and uninfected classes, was utilized for model training and evaluation. Performance evaluation metrics such as precision, recall, F1-score, and accuracy were employed to assess the efficacy of each CNN model in malaria classification.&#x0D; RESULTS: The results demonstrate high accuracy across all models, with AlexNet and VGG19 exhibiting the highest levels of accuracy. However, the selection of a model should consider specific application requirements and constraints, as each model presents unique trade-offs between computational efficiency and performance.&#x0D; CONCLUSION: This study contributes to the burgeoning field of deep learning in healthcare, particularly in utilizing medical imaging for disease diagnosis. The findings underscore the considerable potential of CNNs in enhancing malaria diagnosis. Future research directions may involve further model optimization, exploration of larger and more diverse datasets, and the integration of CNNs into practical diagnostic tools for real-world deployment.

Supporting Malaria Diagnosis Using Deep Learning and Data Augmentation.

Malaria is an infection caused by the Plasmodium parasite that has a major epidemiological, social, and economic impact worldwide. Conventional diagnosis of the disease is based on microscopic examination of thick blood smears. This analysis can be time-consuming, which is key to generate prevention strategies and adequate treatment to avoid the complications associated with the disease. To address this problem, we propose a deep learning-based approach to detect not only malaria parasites but also leukocytes to perform parasite/μL blood count. We used positive and negative images with parasites and leukocytes. We performed data augmentation to increase the size of the dataset. The YOLOv8 algorithm was used for model training and using the counting formula the parasites were counted. The results showed the ability of the model to detect parasites and leukocytes with 95% and 98% accuracy, respectively. The time spent by the model to report parasitemia is significantly less than the time spent by malaria experts. This type of system would be supportive for areas with poor access to health care. We recommend validation of such approaches on a large scale in health institutions.

An aquaporin and an aquaglyceroporin have roles in low temperature adaptation of mosquitoes (Anopheles sinensis).

Mosquitoes (Anopheles sinensis), widely geographically distributed in Asia including China, are the primary vector of the malaria parasite Plasmodium vivax and other parasitic diseases such as Malayan filariasis. An. sinensis can survive through low winter temperatures. Aquaporin channels are found in all life forms, where they facilitate environmental adaptation by allowing rapid trans-cellular movement of water (classical aquaporins) or water and solutes such as glycerol (aquaglyceroporins). Here, we identified and characterized 2 aquaporin (AQP) homologs in An. sinensis: AsAQP2 (An. sinensis aquaglyceroporin) and AsAQP4 (An. sinensis aquaporin). When expressed in frog (Xenopus laevis) oocytes, AsAQP2 transported water, glycerol, and urea; AsAQP4 transported only water. Water permeation through AsAQP2 and AsAQP4 was inhibited by mercuric chloride. AsAQP2 expression was slightly higher in adult female mosquitoes than in males, and AsAQP4 expression was significantly higher in adult males. The 2 AsAQPs were highly expressed in Malpighian tubules and midgut. AsAQP2 and AsAQP4 expression was up-regulated by blood feeding compared with sugar feeding. At freezing point (0°C), the AsAQP4 expression level increased and An. sinensis survival time reduced compared with those at normal temperature (26°C). At low temperature (8°C), the AsAQP2 and AsAQP4 expression levels decreased and survival time was significantly longer compared with those at 26°C. These results suggest that AsAQP2 and AsAQP4 have roles in water homeostasis during blood digestion and in low temperature adaptation of A. sinensis. Together, our results show that the 2 AQPs are important for mosquito diuresis after blood feeding and when exposed to low temperatures.