Emergence of behavioural avoidance strategies of malaria vectors in areas of high LLIN coverage in Tanzania.

Despite significant reductions in malaria transmission across Africa since 2000, progress is stalling. This has been attributed to the development of insecticide resistance and behavioural adaptations in malaria vectors. Whilst insecticide resistance has been widely investigated, there is poorer understanding of the emergence, dynamics and impact of mosquito behavioural adaptations. We conducted a longitudinal investigation of malaria vector host choice over 3 years and resting behaviour over 4 years following a mass long-lasting insecticidal nets (LLINs) distribution in Tanzania. By pairing observations of mosquito ecology with environmental monitoring, we quantified longitudinal shifts in host-choice and resting behaviour that are consistent with adaptation to evade LLINs. The density of An. funestus s.l., declined significantly through time. In tandem, An. arabiensis and An. funestus s.l. exhibited an increased rate of outdoor relative to indoor resting; with An. arabiensis reducing the proportion of blood meals taken from humans in favour of cattle. By accounting for environmental variation, this study detected clear evidence of intra-specific shifts in mosquito behaviour that could be obscured in shorter-term or temporally-coarse surveys. This highlights the importance of mosquito behavioural adaptations to vector control, and the value of longer-term behavioural studies.

Investigating associations between biting time in the malaria vector Anopheles arabiensis Patton and single nucleotide polymorphisms in circadian clock genes: support for sub-structure among An. arabiensis in the Kilombero valley of Tanzania.

BACKGROUND: There is growing evidence that the widespread use of Long-Lasting Insecticidal Nets (LLINs) is prompting malaria vectors to shift their biting towards times and places where people are not protected, such as earlier in the evening and/or outdoors. It is uncertain whether these behavioural shifts are due to phenotypic plasticity and/or ecological changes within vector communities that favour more exophilic species, or involve genetic factors within vector species to limit their contact with LLINs. Possibly variation in the time and location of mosquito biting has a genetic basis, but as yet this phenomenon has received little investigation. Here we used a candidate gene approach to investigate whether polymorphisms in selected circadian clock genes could explain variation in the time and location of feeding (indoors versus outside) within a natural population of the major African malaria vector Anopheles arabiensis. METHODS: Host-seeking An. arabiensis were collected from two villages (Lupiro and Sagamaganga) in Tanzania by Human Landing Catch (HLC) technique. Mosquitoes were classified into phenotypes of "early" (7 pm-10 pm) or "late" biting (4 am -7 am), and host-seeking indoors or outdoors. In these samples we genotyped 34 coding SNPs in 8 clock genes (PER, TIM, CLK, CYC, PDP1, VRI, CRY1, and CRY2), and tested for associations between these SNPs and biting phenotypes. SNPs in 8 mitochondrial genes (ATP6, ATP8, COX1, COX2, COX3, ND3, ND5 and CYTB) were also genotyped to test population subdivision within An. arabiensis. RESULTS: The candidate clock genes exhibited polymorphism within An. arabiensis, but it was unrelated to variation in the timing and location of their biting activity. However, there was evidence of strong genetic structure within An. arabiensis populations in association with the TIM, which was unrelated to geographic distance. Substructure within An. arabiensis was also detected using mitochondrial markers. CONCLUSIONS: The variable timing and location of biting in An. arabiensis could not be linked to candidate clock genes that are known to influence behaviour in other Diptera. This finding does not rule out the possibility of a genetic basis to biting behaviour in this malaria vector, but suggests these are complex phenotypes that require more intensive ecological, neuronal and genomic analyses to understand.

Plasmodium knowlesi transmission: integrating quantitative approaches from epidemiology and ecology to understand malaria as a zoonosis.

The public health threat posed by zoonotic Plasmodium knowlesi appears to be growing: it is increasingly reported across South East Asia, and is the leading cause of malaria in Malaysian Borneo. Plasmodium knowlesi threatens progress towards malaria elimination as aspects of its transmission, such as spillover from wildlife reservoirs and reliance on outdoor-biting vectors, may limit the effectiveness of conventional methods of malaria control. The development of new quantitative approaches that address the ecological complexity of P. knowlesi, particularly through a focus on its primary reservoir hosts, will be required to control it. Here, we review what is known about P. knowlesi transmission, identify key knowledge gaps in the context of current approaches to transmission modelling, and discuss the integration of these approaches with clinical parasitology and geostatistical analysis. We highlight the need to incorporate the influences of fine-scale spatial variation, rapid changes to the landscape, and reservoir population and transmission dynamics. The proposed integrated approach would address the unique challenges posed by malaria as a zoonosis, aid the identification of transmission hotspots, provide insight into the mechanistic links between incidence and land use change and support the design of appropriate interventions.

Disrupted seasonal biology impacts health, food security and ecosystems.

The rhythm of life on earth is shaped by seasonal changes in the environment. Plants and animals show profound annual cycles in physiology, health, morphology, behaviour and demography in response to environmental cues. Seasonal biology impacts ecosystems and agriculture, with consequences for humans and biodiversity. Human populations show robust annual rhythms in health and well-being, and the birth month can have lasting effects that persist throughout life. This review emphasizes the need for a better understanding of seasonal biology against the backdrop of its rapidly progressing disruption through climate change, human lifestyles and other anthropogenic impact. Climate change is modifying annual rhythms to which numerous organisms have adapted, with potential consequences for industries relating to health, ecosystems and food security. Disconcertingly, human lifestyles under artificial conditions of eternal summer provide the most extreme example for disconnect from natural seasons, making humans vulnerable to increased morbidity and mortality. In this review, we introduce scenarios of seasonal disruption, highlight key aspects of seasonal biology and summarize from biomedical, anthropological, veterinary, agricultural and environmental perspectives the recent evidence for seasonal desynchronization between environmental factors and internal rhythms. Because annual rhythms are pervasive across biological systems, they provide a common framework for trans-disciplinary research.

Comparative evaluation of the Sticky-Resting-Box-Trap, the standardised resting-bucket-trap and indoor aspiration for sampling malaria vectors.

BACKGROUND: Understanding mosquito resting behaviour is important for the control of vector-borne diseases, but this remains a challenge because of the paucity of efficient sampling tools. We evaluated two novel sampling methods in the field: the Sticky Resting Box (SRB) and the Resting Bucket trap (RBu) to test their efficiency for sampling malaria vectors resting outdoors and inside houses in rural Tanzania. The performance of RBu and SRB was compared outdoors, while indoors SRB were compared with the Back Pack Aspiration method (BP). Trapping was conducted within 4 villages in the Kilombero Valley, Tanzania over 14 nights. On each night, the performance for collecting Anopheles vectors and Culicinae was compared in 4 households by SRB and RBu outdoors and by SRB or fixed-time Back Pack aspirator in 2 of the 4 focal households indoors. FINDINGS: A total of 619 Anopheles gambiae s.l., 224 Anopheles funestus s.l. and 1737 Culicinae mosquitoes were captured. The mean abundance of An. arabiensis and An. funestus s.l. collected with SRB traps inside and outdoors was significantly lower than with BP or RBu. The SRB however, outperformed BP aspiration for collection of Culicinae indoors. CONCLUSIONS: Of the methods trialled indoors (BP and SRB), BP was the most effective, whilst outdoors RBu performed much better than SRB. However, as SRB can passively sample mosquitoes over a week they could provide an alternative to the RBu where daily monitoring is not possible.

The impact of uniform and mixed species blood meals on the fitness of the mosquito vector Anopheles gambiae s.s: does a specialist pay for diversifying its host species diet?

We investigated the fitness consequences of specialization in an organism whose host choice has an immense impact on human health: the African malaria vector Anopheles gambiae s.s. We tested whether this mosquito's specialism on humans can be attributed to the relative fitness benefits of specialist vs. generalist feeding strategies by contrasting their fecundity and survival on human-only and mixed host diets consisting of blood meals from humans and animals. When given only one blood meal, An. gambiae s.s. survived significantly longer on human and bovine blood, than on canine or avian blood. However, when blood fed repeatedly, there was no evidence that the fitness of An. gambiae s.s. fed a human-only diet was greater than those fed generalist diets. This suggests that the adoption of generalist host feeding strategies in An. gambiae s.s. is not constrained by intraspecific variation in the resource quality of blood from other available host species.

Nature beats nurture: a case study of the physiological fitness of free-living and laboratory-reared male Anopheles gambiae s.l.

Laboratory experimentation forms the basis for most of our knowledge of the biology of many organisms, in particular insects. However, the accuracy with which laboratory-derived estimates of insect life history and behaviour can predict their fitness and population dynamics in the wild is rarely validated. Such comparison is especially important in cases where laboratory-derived information is used to formulate and implement strategies for the genetic control of insects in nature. We have conducted a comparative study of the reproductive potential and life history of male Anopheles gambiae Gilies sensu lato mosquitoes from both standardized laboratory conditions and from natural field settings. We measured three indirect indicators of male mosquito fitness: energetic reserves, body size and survival, in a bid to determine whether the demographics and energetic limitations of wild males can be correctly predicted from their laboratory counterparts. Crucially, the body size and lipid reserves of wild males were substantially greater than those reared under standard laboratory conditions. We caution that the energetic limitations of insects as identified in the laboratory may underestimate their resilience in the wild, and discuss the implications of this phenomenon with respect to vector-borne disease control programmes based on genetic control of mosquitoes.

Plasmodium falciparum malaria disease manifestations in humans and transmission to Anopheles gambiae: a field study in Western Kenya.

Transmission of the malaria parasite Plasmodium is influenced by many different host, vector and parasite factors. Here we conducted a field study at Mbita, an area of endemic malaria in Western Kenya, to test whether parasite transmission to mosquitoes is influenced by the severity of malaria infection in its human host at the time when gametocytes, the transmission forms, are present in the peripheral blood. We examined the infectivity of 81 Plasmodium falciparum gametocyte carriers to mosquitoes. Of these, 21 were patients with fever and other malaria-related symptoms, and 60 were recruited among apparently healthy volunteers. Laboratory-reared Anopheles gambiae s.s. (local strain) were experimentally infected with blood from these gametocyte carriers by membrane-feeding. The severity of the clinical symptoms was greater in febrile patients. These symptomatic patients had higher asexual parasitaemia and lower gametocyte densities (P = 0.05) than healthy volunteers. Ookinete development occurred in only 6 out of the 21 symptomatic patients, of which only 33.3% successfully yielded oocysts. The oocyst prevalence was only 0.6% in the 546 mosquitoes that were fed on blood from this symptomatic group, with mean oocyst intensity of 0.2 (range 0-2) oocysts per mosquito. In contrast, a higher proportion (76.7%) of healthy gametocyte carriers yielded ookinetes, generating an oocyst rate of 12% in the 1332 mosquitoes that fed on them (mean intensity of 6.3, range: 1-105 oocysts per mosquito). Statistical analysis indicated that the increased infectivity of asymptomatic gametocyte carriers was not simply due to their greater gametocyte abundance, but also to the higher level of infectivity of their gametocytes, possibly due to lower parasite mortality within mosquitoes fed on blood from healthy hosts. These results suggest that blood factors and/or conditions correlated with illness reduce P. falciparum gametocyte infectivity.

The energetic budget of Anopheles stephensi infected with Plasmodium chabaudi: is energy depletion a mechanism for virulence?

Evidence continues to accumulate showing that the malaria parasites (Plasmodium spp.) reduce the survival and fecundity of their mosquito vectors (Anopheles spp.). Our ability to identify the possible epidemiological and evolutionary consequences of these parasite-induced fitness reductions has been hampered by a poor understanding of the physiological basis of these shifts. Here, we explore whether the reductions in fecundity and longevity are the result of a parasite-mediated depletion or reallocation of the energetic resources of the mosquito. Mosquitoes infected with Plasmodium chabaudi were expected to have less energetic resources than uninfected mosquitoes, and energy levels were predicted to be lowest in mosquitoes infected with the most virulent parasite genotypes. Not only was there no evidence of a parasite-mediated reduction in the overall energetic budget of mosquitoes, but Plasmodium was actually associated with increased levels of glucose, a key insect nutritional and energetic resource. The data strongly suggest the existence of an increase in sugar feeding in mosquitoes infected with Plasmodium. We suggest different adaptive explanations for an enhanced sugar uptake in infected mosquitoes and call for more studies to investigate the physiological role of glucose in the Plasmodium-mosquito interaction.

The influence of malaria parasite genetic diversity and anaemia on mosquito feeding and fecundity.

Studies of invertebrate-parasite interactions frequently report that infection reduces host fecundity. The extent of the reduction is likely to be determined by a wide range of host and parasite factors. We conducted a laboratory experiment to evaluate the role of parasite genetics and infection genetic diversity on the fecundity of mosquitoes carrying malaria parasites. The malaria vector Anopheles stephensi was infected with either of 2 different genotypes of the rodent malaria parasite Plasmodium chabaudi, or by a mixture of both. Mixed genotype infections reduced mosquito fecundity by 20%, significantly more than either of the 2 single genotype infections. Mixed genotype infections were associated with high gametocyte densities and anaemia in mice, both of which were correlated with reduced bloodmeal size in mosquitoes. Bloodmeal size was the most important predictor of mosquito fecundity; the presence and number of parasites had no direct effect. Parasite density influenced the propensity of mosquitoes to feed on infected mice, with a higher percentage of mosquitoes taking a meal as asexual parasite and gametocyte density increased. Thus mosquitoes may preferentially feed on hosts who will most impair their fecundity.

Mosquito mortality and the evolution of malaria virulence.

Several laboratory studies of malaria parasites (Plasmodium sp.) and some field observations suggest that parasite virulence, defined as the harm a parasite causes to its vertebrate host, is positively correlated with transmission. Given this advantage, what limits the continual evolution of higher parasite virulence? One possibility is that while more virulent strains are more infectious, they are also more lethal to mosquitoes. In this study, we tested whether the virulence of the rodent malaria parasite P. chabaudi in the laboratory mouse was correlated with the fitness of mosquitoes it subsequently infected. Mice were infected with one of seven genetically distinct clones of P. chabaudi that differ in virulence. Weight loss and anemia in infected mice were monitored for 16-17 days before Anopheles stephensi mosquitoes were allowed to take a blood meal from them. Infection virulence in mice was positively correlated with transmission to mosquitoes (infection rate) and weakly associated with parasite burden (number of oocysts). Mosquito survival fell with increasing oocyst burden, but there was no overall statistically significant relationship between virulence in mice and mosquito mortality. Thus, there was no evidence that more virulent strains are more lethal to mosquitoes. Both vector survival and fecundity depended on parasite clone, and contrary to expectations, mosquitoes fed on infections more virulent to mice were more fecund. The strong parasite genetic effects associated with both fecundity and survival suggests that vector fitness could be an important selective agent shaping malaria population genetics and the evolution of phenotypes such as virulence in the vector.

Genetic and environmental determinants of malaria parasite virulence in mosquitoes.

Models of malaria epidemiology and evolution are frequently based on the assumption that vector-parasitic associations are benign. Implicit in this assumption is the supposition that all Plasmodium parasites have an equal and neutral effect on vector survival, and thus that there is no parasite genetic variation for vector virulence. While some data support the assumption of avirulence, there has been no examination of the impact of parasite genetic diversity. We conducted a laboratory study with the rodent malaria parasite, Plasmodium chabaudi and the vector, Anopheles stephensi, to determine whether mosquito mortality varied with parasite genotype (CR and ER clones), infection diversity (single versus mixed genotype) and nutrient availability. Vector mortality varied significantly between parasite genotypes, but the rank order of virulence depended on environmental conditions. In standard conditions, mixed genotype infections were the most virulent but when glucose water was limited, mortality was highest in mosquitoes infected with CR. These genotype-by-environment interactions were repeatable across two experiments and could not be explained by variation in anaemia, gametocytaemia, blood meal size, mosquito body size, infection rate or oocyst burden. Variation in the genetic and environmental determinants of virulence may explain conflicting accounts of Plasmodium pathogenicity to mosquitoes in the malaria literature.

Quality assurance in a Radiation Oncology Unit: the Chart Round experience.

Quality assurance ensures that planned treatments eventuate. Programmes must include feedback loops to promptly correct any shortfall in predetermined standards. In March 1999, a weekly Chart Round was introduced to verify that certain items relevant to quality care were being completed for patients of the Head and Neck Radiotherapy Unit at the Peter MacCallum Cancer Institute. The experience was reviewed after 1 year and it was found that the initiation of Chart Rounds has assisted in raising the level of item completion from 80% to 99% in similar groups of patients treated before and after the initiation of the Chart Round. Initiation of the Chart Round has also provided a useful forum for in-house peer-review, education and effective real-time communication between medical and allied health personnel, all of which has further added to the quality of patient care.

Tumour dose estimation using automated TLD techniques.

Lithium fluoride (TLD-700) dosimeters were used to measure exit surface absorbed doses in external beam radiotherapy using an automated TLD reader. Delivered tumour absorbed doses were derived from these measurements for head and neck, pelvis and breast treatments. For the head and neck treatments (first fraction only), the mean percentage difference between prescribed and delivered tumour absorbed doses was -0.15 +/- 3.0% (+/- 1 SD), for the pelvic treatments -0.83 +/- 2.8% and for the breast treatments +0.26 +/- 2.9%. The spread of results is approximately +/- 3% (+/- 1 SD). This is comparable with the estimated uncertainty in a single TLD absorbed dose measurement in phantom (+/- 2%; +/- 1 SD). Thus, ICRU recommended tolerances for absorbed dose delivery of +/- 5% may not be unequivocally detectable using this method. An action level of +/- 10% is suggested, allowing investigation of possible gross errors in treatment delivery at an early stage, before the course of treatment has progressed to a point at which absorbed dose compensation is impossible.

Automated TLD system for tumor dose estimation from exit dose measurements in external beam radiotherapy.

PURPOSE: An automated TLD facility has been commissioned and calibrated, and techniques have been developed for the measurement of exit doses in external beam radiotherapy, to enable the routine estimation of delivered tumor doses. METHODS AND MATERIALS: An automated TLD system, originally intended for use in diagnostic radiology and radiation protection, has been evaluated and configured for the measurement of exit doses in radiotherapy. Linearity, optimum heating cycles and calibration procedures have been determined. At the photon energies used, encapsulated lithium fluoride chips provide insufficient buildup to ensure electronic equilibrium, necessitating calibration to allow for oblique exit surfaces. Expressions are derived to allow the calculation of delivered tumor doses. RESULTS: Under the calibration conditions described, the uncertainty in a single TLD measurement is approximately +/-2% (+/-1 standard deviation). Over the dose range 0.4-1.5 Gy, TL response is linear. The total heating cycle time, including annealing, is 75 s. Measurements of R(exit) (the ratio of exit dose with and without full backscatter), used in the estimation of tumor doses, decreases with field size for small fields and varies only slightly for field sizes greater than 7 x 7 cm. Lack of electronic equilibrium leads to a decrease in R(exit) with increasing exit surface obliquity for all energies considered. Application of the technique to a simulated treatment showed good agreement between estimated and applied tumor doses, when surface obliquity was taken into account. CONCLUSION: This work describes the commissioning and calibration of an automated TLD facility and demonstrates that exit surface measurements using TLD chips used under conditions where electronic equilibrium was not established, have the potential for identifying discrepancies in delivered tumor doses.

Antarctica: measuring glacier velocity from satellite images.

Many Landsat images of Antarctica show distinctive flow and crevasse features in the floating part of ice streams and outlet glaciers immediately below their grounding zones. Some of the features, which move with the glacier or ice stream, remain visible over many years and thus allow time-lapse measurements of ice velocities. Measurements taken from Landsat images of features on Byrd Glacier agree well with detailed ground and aerial observations. The satellite-image technique thus offers a rapid and cost-effective method of obtaining average velocities, to a first order of accuracy, of many ice streams and outlet glaciers near their termini.