Estimating the extrinsic incubation period of malaria using a mechanistic model of sporogony

During sporogony, malaria-causing parasites infect a mosquito, reproduce and migrate to the mosquito salivary glands where they can be transmitted the next time blood feeding occurs. The time required for sporogony, known as the extrinsic incubation period (EIP), is an important determinant of malaria transmission intensity. The EIP is typically estimated as the time for a given percentile, x, of infected mosquitoes to develop salivary gland sporozoites (the infectious parasite life stage), which is denoted by EIP_x. Many mechanisms, however, affect the observed sporozoite prevalence including the human-to-mosquito transmission probability and possibly differences in mosquito mortality according to infection status. To account for these various mechanisms, we present a mechanistic mathematical model, which explicitly models key processes at the parasite, mosquito and observational scales. Fitting this model to experimental data, we find greater variation in the EIP than previously thought: we estimated the range between EIP₁₀ and EIP₉₀ (at 27°C) as 4.5 days compared to 0.9 days using existing statistical methods. This pattern holds over the range of study temperatures included in the dataset. Increasing temperature from 21°C to 34°C decreased the EIP₅₀ from 16.1 to 8.8 days. Our work highlights the importance of mechanistic modelling of sporogony to (1) improve estimates of malaria transmission under different environmental conditions or disease control programs and (2) evaluate novel interventions that target the mosquito life stages of the parasite.     

A mathematical model for zoonotic transmission of malaria in the Atlantic Forest: Exploring the effects of variations in vector abundance and acrodendrophily

Transmission foci of autochthonous malaria caused by Plasmodium vivax-like parasites have frequently been reported in the Atlantic Forest in Southeastern and Southern Brazil. Evidence suggests that malaria is a zoonosis in these areas as human infections by simian Plasmodium species have been detected, and the main vector of malaria in the Atlantic Forest, Anopheles (Kerteszia) cruzii, can blood feed on human and simian hosts. In view of the lack of models that seek to predict the dynamics of zoonotic transmission in this part of the Atlantic Forest, the present study proposes a new deterministic mathematical model that includes a transmission compartment for non-human primates and parameters that take into account vector displacement between the upper and lower forest strata. The effects of variations in the abundance and acrodendrophily of An. cruzii on the prevalence of infected humans in the study area and the basic reproduction number (R₀) for malaria were analyzed. The model parameters are based on the literature and fitting of the empirical data. Simulations performed with the model indicate that (1) an increase in the abundance of the vector in relation to the total number of blood-seeking mosquitoes leads to an asymptotic increase in both the proportion of infected individuals at steady state and R₀; (2) the proportion of infected humans at steady state is higher when displacement of the vector mosquito between the forest strata increases; and (3) in most scenarios, Plasmodium transmission cannot be sustained only between mosquitoes and humans, which implies that non-human primates play an important role in maintaining the transmission cycle. The proposed model contributes to a better understanding of the dynamics of malaria transmission in the Atlantic Forest.     

Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.     

Malaria Vector Surveillance in Ganghwa-do, a Malaria-Endemic Area in the Republic of Korea

We investigated the seasonality of Anopheles mosquitoes, including its species composition, density, parity, and population densities of mosquitoes infected with the parasite in Ganghwa-do (Island), a vivax malaria endemic area in the Republic of Korea. Mosquitoes were collected periodically with a dry-ice-tent trap and a blacklight trap during the mosquito season (April-October) in 2008. Anopheles sinensis (94.9%) was the most abundant species collected, followed by Anopheles belenrae (3.8%), Anopheles pullus (1.2%), and Anopheles lesteri (0.1%). Hibernating Anopheles mosquitoes were also collected from December 2007 to March 2008. An. pullus (72.1%) was the most frequently collected, followed by An. sinensis (18.4%) and An. belenrae (9.5%). The composition of Anopheles species differed between the mosquito season and hibernation seasons. The parous rate fluctuated from 0% to 92.9%, and the highest rate was recorded on 10 September 2008. Sporozoite infections were detected by PCR in the head and thorax of female Anopheles mosquitoes. The annual sporozoite rate of mosquitoes was 0.11% (2 of 1,845 mosquitoes). The 2 mosquitoes that tested positive for sporozoites were An. sinensis. Malarial infections in anopheline mosquitoes from a population pool were also tried irrespective of the mosquito species. Nine of 2,331 pools of Anopheles mosquitoes were positive. From our study, it can be concluded that An. sinensis, which was the predominant vector species and confirmed as sporozoite-infected, plays an important role in malaria transmission in Ganghwa-do.     

A weather-driven model of malaria transmission

The classic formulae in malaria epidemiology are reviewed that relate entomological parameters to malaria transmission, including mosquito survivorship and age-at-infection, the stability index (S), the human blood index (HBI), proportion of infected mosquitoes, the sporozoite rate, the entomological inoculation rate (EIR), vectorial capacity (C) and the basic reproductive number (R ₀). The synthesis emphasizes the relationships among classic formulae and reformulates a simple dynamic model for the proportion of infected humans. The classic formulae are related to formulae from cyclical feeding models, and some inconsistencies are noted. The classic formulae are used to to illustrate how malaria control reduces malaria transmission and show that increased mosquito mortality has an effect even larger than was proposed by Macdonald in the 1950's.     

Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential

Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite’s extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R₀, and find the average R₀ is higher (range: 10.1%–12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions.     

Attraction of mosquitoes to primate odours and implications for zoonotic Plasmodium transmission

Vector-borne diseases often originate from wildlife and can spill over into the human population. One of the most important determinants of vector-borne disease transmission is the host preference of mosquitoes. Mosquitoes with a specialised host preference are guided by body odours to find their hosts in addition to carbon dioxide. Little is known about the role of mosquito host preference in the spillover of pathogenic agents from humans towards animals and vice versa. In the Republic of Congo, the attraction of mosquitoes to primate host odours was determined, as well as their possible role as malaria vectors, using odour-baited traps mimicking the potential hosts of mosquitoes. Most of the mosquito species caught showed a generalistic host preference. Anopheles obscurus was the most abundant Anopheles mosquito, with a generalistic host preference observed from the olfactory response and the detection of various Plasmodium parasites. Interestingly, Culex decens showed a much higher attraction towards chimpanzee odours than to human or cow odours. Human Plasmodium parasites were observed in both human and chimpanzee blood, although not in the Anopheles mosquitoes that were collected. Understanding the role of mosquito host preference for cross-species parasite transmission provides information that will help to determine the risk of spillover of vector-borne diseases.     

Long-Lasting Insecticidal Nets Are Synergistic with Mass Drug Administration for Interruption of Lymphatic Filariasis Transmission in Nigeria

In central Nigeria Anopheles mosquitoes transmit malaria and lymphatic filariasis (LF). The strategy used for interrupting LF transmission in this area is annual mass drug administration (MDA) with albendazole and ivermectin, but after 8 years of MDA, entomological evaluations in sentinel villages showed continued low-grade mosquito infection rates of 0.32%. After long-lasting insecticidal net (LLIN) distribution by the national malaria program in late 2010, however, we were no longer able to detect infected vectors over a 24-month period. This is evidence that LLINs are synergistic with MDA in interrupting LF transmission.     

Examining Landscape Factors Influencing Relative Distribution of Mosquito Genera and Frequency of Virus Infection

Mosquito-borne infections cause some of the most debilitating human diseases, including yellow fever and malaria, yet we lack an understanding of how disease risk scales with human-driven habitat changes. We present an approach to study variation in mosquito distribution and concomitant viral infections on the landscape level. In a pilot study we analyzed mosquito distribution along a 10-km transect of a West African rainforest area, which included primary forest, secondary forest, plantations, and human settlements. Variation was observed in the abundance of Anopheles, Aedes, Culex, and Uranotaenia mosquitoes between the different habitat types. Screening of trapped mosquitoes from the different habitats led to the isolation of five uncharacterized viruses of the families Bunyaviridae, Coronaviridae, Flaviviridae, and Rhabdoviridae, as well as an unclassified virus. Polymerase chain reaction screening for these five viruses in individual mosquitoes indicated a trend toward infection with specific viruses in specific mosquito genera that differed by habitat. Based on these initial analyses, we believe that further work is indicated to investigate the impact of anthropogenic landscape changes on mosquito distribution and accompanying arbovirus infection.     

ANOSPEX: A Stochastic,Spatially Explicit Model for Studying Anopheles Metapopulation Dynamics

Anopheles mosquitoes transmit malaria, a major public health problem among many African countries. One of the most effective methods to control malaria is by controlling the Anopheles mosquito vectors that transmit the parasites. Mathematical models have both predictive and explorative utility to investigate the pros and cons of different malaria control strategies. We have developed a C++ based, stochastic spatially explicit model (ANOSPEX; Ano phelesSpatially-Explicit) to simulate Anopheles metapopulation dynamics. The model is biologically rich, parameterized by field data, and driven by field-collected weather data from Macha, Zambia. To preliminarily validate ANOSPEX, simulation results were compared to field mosquito collection data from Macha; simulated and observed dynamics were similar. The ANOSPEX model will be useful in a predictive and exploratory manner to develop, evaluate and implement traditional and novel strategies to control malaria, and for understanding the environmental forces driving Anopheles population dynamics.     

Malaria vectors of Papua New Guinea

Understanding malaria transmission in Papua New Guinea (PNG) requires exact knowledge of which Anopheles species are transmitting malaria and is complicated by the cryptic species status of many of these mosquitoes. To identify the malaria vectors in PNG we studied Anopheles specimens from 232 collection localities around human habitation throughout PNG (using CO₂ baited light traps and human bait collections). A total of 22,970 mosquitoes were individually assessed using a Plasmodium sporozoite enzyme-linked immunosorbent assay to identify Plasmodiumfalciparum, Plasmodiumvivax and Plasmodiummalariae circumsporozoite proteins. All mosquitoes were identified to species by morphology and/or PCR. Based on distribution, abundance and their ability to develop sporozoites, we identified five species as major vectors of malaria in PNG. These included: Anophelesfarauti, Anopheleshinesorum (incriminated here, to our knowledge, for the first time), Anophelesfarauti 4, Anopheleskoliensis and Anophelespunctulatus. Anopheleslongirostris and Anophelesbancroftii were also incriminated in this study. Surprisingly, An. longirostris showed a high incidence of infections in some areas. A newly identified taxon within the Punctulatus Group, tentatively called An. farauti 8, was also found positive for circumsporozoite protein. These latter three species, together with Anopheleskarwari and Anophelessubpictus, incriminated in other studies, appear to be only minor vectors, while Anophelesfarauti 6 appears to be the major vector in the highland river valleys (>1500 m above sea level). The nine remaining Anopheles species found in PNG have been little studied and their bionomics are unknown; most appear to be uncommon with limited distribution and their possible role in malaria transmission has yet to be determined.     

Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.     

Effect of irrigation systems on temporal distribution of malaria vectors in semi-arid regions

Previous research models have used climate data to explain habitat conditions of Anopheles mosquitoes transmitting malaria parasites. Although they can estimate mosquito populations with sufficient accuracy in many areas, observational data show that there is a tendency to underestimate the active growth and reproduction period of mosquitoes in semi-arid agricultural regions. In this study, a new, modified model that includes irrigation as a factor was developed to predict the active growing period of mosquitoes more precisely than the base model for ecophysiological and climatological distribution of mosquito generations (ECD-mg). Five sites with complete sets of observational data were selected in semi-arid regions of India for the comparison. The active growing period of mosquitoes determined from the modified ECD-mg model that incorporated the irrigation factor was in agreement with the observational data, whereas the active growing period was underestimated by the previous ECD-mg model that did not incorporate irrigation. This suggests that anthropogenic changes in the water supply due to extensive irrigation can encourage the growth of Anopheles mosquitoes through the alteration of the natural water balance in their habitat. In addition, it was found that the irrigation systems not only enable the active growth of mosquitoes in dry seasons but also play an important role in stabilizing the growth in rainy seasons. Consequently, the irrigation systems could lengthen the annual growing period of Anopheles mosquitoes and increase the maximum generation number of mosquitoes in semi-arid subtropical regions.     

How the Malaria Vector Anopheles gambiae Adapts to the Use of Insecticide-Treated Nets by African Populations

BackgroundInsecticide treated bed nets have been recommended and proven efficient as a measure to protect African populations from malaria mosquito vector Anopheles spp. This study evaluates the consequences of bed nets use on vectors resistance to insecticides, their feeding behavior and malaria transmission in Dielmo village, Senegal, were LLINs were offered to all villagers in July 2008.MethodsAdult mosquitoes were collected monthly from January 2006 to December 2011 by human landing catches (HLC) and by pyrethroid spray catches (PCS). A randomly selected sub-sample of 15–20% of An. gambiae s.l. collected each month was used to investigate the molecular forms of the An. gambiae complex, kdr mutations, and Plasmodium falciparum circumsporozoite (CSP) rate. Malaria prevalence and gametocytaemia in Dielmo villagers were measured quarterly.ResultsInsecticide susceptible mosquitoes (wild kdr genotype) presented a reduced lifespan after LLINs implementation but they rapidly adapted their feeding behavior, becoming more exophageous and zoophilic, and biting earlier during the night. In the meantime, insecticide-resistant specimens (kdr L1014F genotype) increased in frequency in the population, with an unchanged lifespan and feeding behaviour. P. falciparum prevalence and gametocyte rate in villagers decreased dramatically after LLINs deployment. Malaria infection rate tended to zero in susceptible mosquitoes whereas the infection rate increased markedly in the kdr homozygote mosquitoes.ConclusionDramatic changes in vector populations and their behavior occurred after the deployment of LLINs due to the extraordinary adaptative skills of An. gambiae s. l. mosquitoes. However, despite the increasing proportion of insecticide resistant mosquitoes and their almost exclusive responsibility in malaria transmission, the P. falciparum gametocyte reservoir continued to decrease three years after the deployment of LLINs.     

An inventory of human night-biting mosquitoes and their bionomics in Sumba,Indonesia

Mosquitoes are important vectors that transmit pathogens to human and other vertebrates. Each mosquito species has specific ecological requirements and bionomic traits that impact human exposure to mosquito bites, and hence disease transmission and vector control. A study of human biting mosquitoes and their bionomic characteristics was conducted in West Sumba and Southwest Sumba Districts, Nusa Tenggara Timur Province, Indonesia from May 2015 to April 2018. Biweekly human landing catches (HLC) of night biting mosquitoes both indoors and outdoors caught a total of 73,507 mosquito specimens (59.7% non-Anopheles, 40.3% Anopheles). A minimum of 22 Culicinae species belonging to four genera (Aedes, Armigeres, Culex, Mansonia), and 13 Anophelinae species were identified. Culex quinquefasciatus was the dominant Culicinae species, Anopheles aconitus was the principal Anopheles species inland, while An. sundaicus was dominant closer to the coast. The overall human biting rate (HBR) was 10.548 bites per person per night (bpn) indoors and 10.551 bpn outdoors. Mosquitoes biting rates were slightly higher indoors for all genera with the exception of Anopheles, where biting rates were slightly higher outdoors. Diurnal and crepuscular Aedes and Armigeres demonstrated declining biting rates throughout the night while Culex and Anopheles biting rates peaked before midnight and then declined. Both anopheline and non-anopheline populations did not have a significant association with temperature (p = 0.3 and 0.88 respectively), or rainfall (p = 0.13 and 0.57 respectively). The point distribution of HBR and seasonal variables did not have a linear correlation. Data demonstrated similar mosquito–human interactions occurring outdoors and indoors and during early parts of the night implying both indoor and outdoor disease transmission potential in the area–pointing to the need for interventions in both spaces. Integrated vector analysis frameworks may enable better surveillance, monitoring and evaluation strategies for multiple diseases.     

Transgenic Mosquitoes Expressing a Phospholipase A2 Gene Have a Fitness Advantage When Fed Plasmodium falciparum-Infected Blood

Background

Genetically modified mosquitoes have been proposed as an alternative strategy to reduce the heavy burden of malaria. In recent years, several proof-of-principle experiments have been performed that validate the idea that mosquitoes can be genetically modified to become refractory to malaria parasite development.

Results

We have created two transgenic lines of Anopheles stephensi , a natural vector of Plasmodium falciparum, which constitutively secrete a catalytically inactive phospholipase A₂ (mPLA₂) into the midgut lumen to interfere with Plasmodium ookinete invasion. Our experiments show that both transgenic lines expressing mPLA₂ significantly impair the development of rodent malaria parasites, but only one line impairs the development of human malaria parasites. In addition, when fed on malaria-infected blood, mosquitoes from both transgenic lines are more fecund than non-transgenic mosquitoes. Consistent with these observations, cage experiments with mixed populations of transgenic and non-transgenic mosquitoes show that the percentage of transgenic mosquitoes increases when maintained on Plasmodium -infected blood.

Conclusions

Our results suggest that the expression of an anti-Plasmodium effector gene gives transgenic mosquitoes a fitness advantage when fed malaria-infected blood. These findings have important implications for future applications of transgenic mosquito technology in malaria control.     

Transgenic technologies to induce sterility

The last few years have witnessed a considerable expansion in the number of tools available to perform molecular and genetic studies on the genome of Anopheles mosquitoes, the vectors of human malaria. As a consequence, knowledge of aspects of the biology of mosquitoes, such as immunity, reproduction and behaviour, that are relevant to their ability to transmit disease is rapidly increasing, and could be translated into concrete benefits for malaria control strategies. Amongst the most important scientific advances, the development of transgenic technologies for Anopheles mosquitoes provides a crucial opportunity to improve current vector control measures or design novel ones. In particular, the use of genetic modification of the mosquito genome could provide for a more effective deployment of the sterile insect technique (SIT) against vector populations in the field. Currently, SIT relies on the release of radiation sterilized males, which compete with wild males for mating with wild females. The induction of sterility in males through the genetic manipulation of the mosquito genome, already achieved in a number of other insect species, could eliminate the need for radiation and increase the efficiency of SIT-based strategies. This paper provides an overview of the mechanisms already in use for inducing sterility by transgenesis in Drosophila and other insects, and speculates on possible ways to apply similar approaches to Anopheles mosquitoes.     

Plasmodium spp.: dispersion of malarial oocyst populations in anopheline and culicine mosquitoes

The numbers of malarial oocysts developing in individual, like mosquitoes fed concurrently on a single vertebrate malarial host were found to be distributed according to the negative binomial distribution in 169 experiments utilizing 6 species of Plasmodium, 6 species of mosquitoes and 3 species of vertebrate hosts. Dispersion constants ranged upward to 8.0, and mean clump sizes ranged upward to 298.4. The dispersion constant was demonstrated to be contingent on the species, strain and identity of the mosquito, the parasite and the vertebrate host; on the genetic state of the mosquito; and on the state of the infection in the vertebrate host. It was concluded that the concentration of oocyst production in particular mosquitoes was produced by varying levels and combinations of numerous factors associated with the parasite, the mosquito and the vertebrate host and that the pattern of oocyst distribution favors parasite survival and the maintenance of malaria in the field.     

Anopheline species and their Plasmodium infection status in Aligarh,India

Malaria is a global issue and India contributes substantially to global malaria incidence. Information related to malaria vectors is very limited in Aligarh. The environmental and climatological situations permit the continual breeding of vectors in permanent breeding sites. This study was designed with the aim to screen all the anophelines species and possible malaria vectors in three different localities of Aligarh. Anopheles mosquitoes were collected from three different localities (Fort, Jalali and Tappal) during peak malaria transmission season (July to November) by using mouth aspirator and CDC light traps. Enzyme-linked immunosorbent assay (ELISA) was done to detect Plasmodium falciparum, Plasmodium vivax-210 and P. vivax-247 circumsporozoite proteins (CSP) from the collected female species. A total of 794 female anopheline mosquitoes belonging to 7 species were collected by different methods. Circumsporozoite protein–enzyme-linked immunosorbent assay was performed with 780 anopheline mosquitoes out of which 13 mosquitoes were positive in CSP–ELISA. Thus, the overall infection rate was 1.66% (13/780). Four (0.51%) mosquitoes belonging to three species were positive for P. falciparum, 7 (0.89%) mosquitoes belonging to three species were positive for VK 210 and 2 (0.25%) mosquitoes belonging to Anopheles culicifacies and Anopheles stephensi species were positive for VK 247. No mixed infection was found in this study. According to species, the highest infection rate was observed in An. culicifacies (7/288, 2.43%) followed by An. stephensi (2.40%) and Anopheles annularis (1.98%). An. culicifacies and An. stephensi were previously incriminated as malaria vectors in Aligarh. There was, however, no previous report in favor of infections in An. annularis in Aligarh. The on-going Malaria Control Program in India needs up to date information on malaria vectors. A major challenge is the lack of knowledge about vectors and their role in malaria transmission. Findings of this study suggested that in the absence of major malaria vectors there is a possibility that other Anopheles species may have been playing a role in malaria transmission in Aligarh.