Plasmodium relictum infection in Culex quinquefasciatus (Culicidae) decreases diel flight activity but increases peak dusk flight activity.

BACKGROUND: Parasites are recognized for their ability to modify host physiology and behaviours in ways that increase parasite fitness. Protozoan parasites of the genus Plasmodium are a group of widespread vector-borne parasites of vertebrates, causing disease to a wide range of hosts, but most notably to human and avian hosts. METHODS: The hypothesis that infection with the avian malaria, Plasmodium relictum (GRW4 lineage) impacts flight activity in one of their natural vectors, Culex quinquefasciatus, was tested using both parasites and mosquitoes colonized from local populations in East-Central Texas, USA. Groups of Cx. quinquefasciatus were allowed to feed directly on canaries with active P. relictum infections and control canaries with no P. relictum exposure history. Additionally, how P. relictum sporozoite invasion of mosquito salivary glands impacts mosquito flight activity behaviour was tested using a Locomotor Activity Monitor for both control and infected females. Generalized linear mixed models were used to evaluate the influence of infection status on the response variables of flight activity (continuous) and probability of flight occurring (binomial). RESULTS: Infection status was a significant predictor of flight activity and flight probability and interactions between infection status and experimental period of infection as well as infection status and dusk were statistically significant predictors of flight activity. Plasmodium relictum infected mosquitoes had a mean flight activity of 3.10 and control mosquitoes had an overall mean flight activity of 3.13. DISCUSSION: Based on these results, avian malaria parasites increase the flight activity of these mosquitoes at hours known for peak host-seeking behaviour but decrease overall diel activity. CONCLUSION: Although the ramifications of this behavioural change for P. relictum transmission are unclear, these results provide additional empirical evidence suggesting that avian malaria can influence mosquito behaviour and modulate transmission potential.

Impact of disabled circadian clock on yellow fever mosquito Aedes aegypti fitness and behaviors.

Like other insects, Aedes aegypti displays strong daily patterns in host seeking and mating. Much of these behaviors are believed to be under the control of a circadian clock, an endogenous timekeeping mechanism relying on transcriptional/translational negative feedback loops that drive rhythmic physiology and behavior. To examine the connection between the circadian clock and various Ae. aegypti behaviors, we knocked out the core clock gene cycle using CRISPR/Cas9. We found that the rhythmic pattern and intensity of mRNA expression of seven circadian genes, including AeCyc-/-, were altered across the day/night cycle as well as in constant darkness conditions. We further show that the mutant CYC protein is incapable of forming a dimer with CLK to stimulate per expression and that the endogenous clock is disabled in AeCyc-/- mosquitoes. AeCyc-/- do not display the bimodal locomotor activity pattern of wild type, have a significantly reduced response to host odor, reduced egg hatching rates, delayed embryonic development and reduced adult survival and mating success. Surprisingly however, the propensity to blood feed in AeCyc-/- females is significantly higher than in wildtype females. Together with other recent work on the circadian clock control of key aspects of mosquito biology, our data on how cycle KO affects mosquito behavior and fitness provides a basis for further work into the pathways that connect the mosquito endogenous clock to its vector competence.

Increased Biting Rate of Insecticide-Resistant Culex Mosquitoes and Community Adherence to IRS for Malaria Control in Urban Malabo, Bioko Island, Equatorial Guinea.

Sustaining high levels of indoor residual spraying (IRS) coverage (≥85%) for community protection against malaria remains a challenge for IRS campaigns. We examined biting rates and insecticide resistance in Culex species and Anopheles gambiae s.l., and their potential effect on community adherence to IRS. The average IRS coverage in urban Malabo between 2015 and 2017 remained at 80%. Culex biting rate increased 6.0-fold (P < 0.001) between 2014 and 2017, reaching 8.08 bites per person per night, whereas that of An. gambiae s.l. remained steady at around 0.68. Although An. gambiae s.l. was susceptible to carbamates and organophosphates insecticides, Culex spp. were phenotypically resistant to all four main classes of WHO-recommended IRS insecticides. Similarly, the residual activity of the organophosphate insecticide used since 2017, ACTELLIC 300CS, was 8 mo for An. gambiae s.l., but was almost absent against Culex for 2 mo post-spray. A survey conducted in 2018 within urban Malabo indicated that 77.0% of respondents related IRS as means of protection against mosquito bites, but only 3.2% knew that only Anopheles mosquitoes transmit malaria. Therefore, the increasing biting rates of culicines in urban Malabo, and their resistance to all IRS insecticides, is raising concern that a growing number of people may refuse to participate in IRS as result of its perceived failure in controlling mosquitoes. Although this is not yet the case on Bioko Island, communication strategies need refining to sensitize communities about the effectiveness of IRS in controlling malaria vectors in the midst of insecticide resistance in nonmalaria vector mosquitoes.

Analysis of near infrared spectra for age-grading of wild populations of Anopheles gambiae.

BACKGROUND: Understanding the age-structure of mosquito populations, especially malaria vectors such as Anopheles gambiae, is important for assessing the risk of infectious mosquitoes, and how vector control interventions may impact this risk. The use of near-infrared spectroscopy (NIRS) for age-grading has been demonstrated previously on laboratory and semi-field mosquitoes, but to date has not been utilized on wild-caught mosquitoes whose age is externally validated via parity status or parasite infection stage. In this study, we developed regression and classification models using NIRS on datasets of wild An. gambiae (s.l.) reared from larvae collected from the field in Burkina Faso, and two laboratory strains. We compared the accuracy of these models for predicting the ages of wild-caught mosquitoes that had been scored for their parity status as well as for positivity for Plasmodium sporozoites. RESULTS: Regression models utilizing variable selection increased predictive accuracy over the more common full-spectrum partial least squares (PLS) approach for cross-validation of the datasets, validation, and independent test sets. Models produced from datasets that included the greatest range of mosquito samples (i.e. different sampling locations and times) had the highest predictive accuracy on independent testing sets, though overall accuracy on these samples was low. For classification, we found that intramodel accuracy ranged between 73.5-97.0% for grouping of mosquitoes into "early" and "late" age classes, with the highest prediction accuracy found in laboratory colonized mosquitoes. However, this accuracy was decreased on test sets, with the highest classification of an independent set of wild-caught larvae reared to set ages being 69.6%. CONCLUSIONS: Variation in NIRS data, likely from dietary, genetic, and other factors limits the accuracy of this technique with wild-caught mosquitoes. Alternative algorithms may help improve prediction accuracy, but care should be taken to either maximize variety in models or minimize confounders.

Increasing outdoor host-seeking in Anopheles gambiae over 6 years of vector control on Bioko Island.

BACKGROUND: Vector control through indoor residual spraying (IRS) has been employed on Bioko Island, Equatorial Guinea, under the Bioko Island Malaria Control Project (BIMCP) since 2004. This study analyses the change in mosquito abundance, species composition and outdoor host-seeking proportions from 2009 to 2014, after 11 years of vector control on Bioko Island. METHODS: All-night indoor and outdoor human landing catches were performed monthly in the Bioko Island villages of Mongola, Arena Blanca, Biabia and Balboa from 2009 to 2014. Collected mosquitoes were morphologically identified and a subset of Anopheles gambiae sensu lato (s.l.) were later identified molecularly to their sibling species. Mosquito collection rates, species composition and indoor/outdoor host-seeking sites were analysed using generalized linear mixed models to assess changes in mosquito abundance and behaviour. RESULTS: The overall mosquito collection rate declined in each of the four Bioko Island villages. Anopheles coluzzii and Anopheles melas comprised the An. gambiae s.l. mosquito vector population, with a range of species proportions across the four villages. The proportion of outdoor host-seeking An. gambiae s.l. mosquitoes increased significantly in all four villages with an average increase of 58.8 % [57.9, 59.64 %] in 2009 to 70.0 % [67.8, 72.0 %] in 2014. Outdoor host-seeking rates did not increase in the month after an IRS spray round compared to the month before, suggesting that insecticide repellency has little impact on host-seeking behaviour. CONCLUSION: While vector control on Bioko Island has succeeded in substantial reduction in overall vector biting rates, populations of An. coluzzii and An. melas persist. Host-seeking behaviour has changed in these An. gambiae s.l. populations, with a shift towards outdoor host-seeking. During this study period, the proportion of host-seeking An. gambiae s.l. caught outdoors observed on Bioko Island increased to high levels, exceeding 80 % in some locations. It is possible that there may be a genetic basis underlying this large shift in host-seeking behaviour, in which case outdoor feeding could pose a serious threat to current vector control programmes. Currently, the BIMCP is preparing for this potential challenge by testing source reduction as a complementary control effort that also targets outdoor transmission.

Age and prior blood feeding of Anopheles gambiae influences their susceptibility and gene expression patterns to ivermectin-containing blood meals.

BACKGROUND: Ivermectin has been proposed as a novel malaria transmission control tool based on its insecticidal properties and unique route of acquisition through human blood. To maximize ivermectin's effect and identify potential resistance/tolerance mechanisms, it is important to understand its effect on mosquito physiology and potential to shift mosquito population age-structure. We therefore investigated ivermectin susceptibility and gene expression changes in several age groups of female Anopheles gambiae mosquitoes. METHODS: The effect of aging on ivermectin susceptibility was analyzed in three age groups (2, 6, and 14-days) of colonized female Anopheles gambiaemosquitoes using standard survivorship assays. Gene expression patterns were then analyzed by transcriptome sequencing on an Illumina HiSeq 2500 platform. RT-qPCR was used to validate transcriptional changes and also to examine expression in a different, colonized strain and in wild mosquitoes, both of which blood fed naturally on an ivermectin-treated person. RESULTS: Mosquitoes of different ages and blood meal history died at different frequencies after ingesting ivermectin. Mortality was lowest in 2-day old mosquitoes exposed on their first blood meal and highest in 6-day old mosquitoes exposed on their second blood meal. Twenty-four hours following ivermectin ingestion, 101 and 187 genes were differentially-expressed relative to control blood-fed, in 2 and 6-day groups, respectively. Transcription patterns of select genes were similar in membrane-fed, colonized, and naturally-fed wild vectors. Transcripts from several unexpected functional classes were highly up-regulated, including Niemann-Pick Type C (NPC) genes, peritrophic matrix-associated genes, and immune-response genes, and these exhibited different transcription patterns between age groups, which may explain the observed susceptibility differences. Niemann-Pick Type 2 genes were the most highly up-regulated transcripts after ivermectin ingestion (up to 160 fold) and comparing phylogeny to transcriptional patterns revealed that NPCs have rapidly evolved and separate members respond to either blood meals or to ivermectin. CONCLUSION: We present evidence of increased ivermectin susceptibility in older An. gambiae mosquitoes that had previously bloodfed. Differential expression analysis suggests complex midgut interactions resulting from ivermectin ingestion that likely involve blood meal digestion physiological responses, midgut microflora, and innate immune responses. Thus, the transcription of certain gene families is consistently affected by ivermectin ingestion, and may provide important clues to ivermectin's broad effects on malaria vectors. These findings contribute to the growing understanding of ivermectin's potential as a transmission control tool.

Characterization of the target of ivermectin, the glutamate-gated chloride channel, from Anopheles gambiae.

The use of insecticide-treated nets and indoor residual insecticides targeting adult mosquito vectors is a key element in malaria control programs. However, mosquito resistance to the insecticides used in these applications threatens malaria control efforts. Recently, the mass drug administration of ivermectin (IVM) has been shown to kill Anopheles gambiae mosquitoes and disrupt Plasmodium falciparum transmission in the field. We cloned the molecular target of IVM from A. gambiae, the glutamate-gated chloride channel (AgGluCl), and characterized its transcriptional patterns, protein expression and functional responses to glutamate and IVM. AgGluCl cloning revealed an unpredicted fourth splice isoform as well as a novel exon and splice site. The predicted gene products contained heterogeneity in the N-terminal extracellular domain and the intracellular loop region. Responses to glutamate and IVM were measured using two-electrode voltage clamp on Xenopus laevis oocytes expressing AgGluCl. IVM induced non-persistent currents in AgGluCl-a1 and did not potentiate glutamate responses. In contrast, AgGluCl-b was insensitive to IVM, suggesting that the AgGluCl gene could produce IVM-sensitive and -insensitive homomultimers from alternative splicing. AgGluCl isoform-specific transcripts were measured across tissues, ages, blood feeding status and sex, and were found to be differentially transcribed across these physiological variables. Lastly, we stained adult, female A. gambiae for GluCl expression. The channel was expressed in the antenna, Johnston's organ, supraesophageal ganglion and thoracic ganglia. In summary, we have characterized the first GluCl from a mosquito, A. gambiae, and described its unique activity and expression with respect to it as the target of the insecticide IVM.

Mosquitocidal properties of IgG targeting the glutamate-gated chloride channel in three mosquito disease vectors (Diptera: Culicidae).

The glutamate-gated chloride channel (GluCl) is a highly sensitive insecticide target of the avermectin class of insecticides. As an alternative to using chemical insecticides to kill mosquitoes, we tested the effects of purified immunoglobulin G (IgG) targeting the extracellular domain of GluCl from Anopheles gambiae (AgGluCl) on the survivorship of three key mosquito disease vectors: Anopheles gambiae s.s., Aedes aegypti and Culex tarsalis. When administered through a single blood meal, anti-AgGluCl IgG reduced the survivorship of A. gambiae in a dose-dependent manner (LC50: 2.82 mg ml(-1), range 2.68-2.96 mg ml(-1)) but not A. aegypti or C. tarsalis. We previously demonstrated that AgGluCl is only located in tissues of the head and thorax of A. gambiae. To verify that AgGluCl IgG is affecting target antigens found outside the midgut, we injected it directly into the hemocoel via intrathoracic injection. A single, physiologically relevant concentration of anti-AgGluCl IgG injected into the hemocoel equally reduced mosquito survivorship of all three species. To test whether anti-AgGluCl IgG was entering the hemocoel of each of these mosquitoes, we fed mosquitoes a blood meal containing anti-AgGluCl IgG and subsequently extracted their hemolymph. We only detected IgG in the hemolymph of A. gambiae, suggesting that resistance of A. aegypti and C. tarsalis to anti-AgGluCl IgG found in blood meals is due to deficient IgG translocation across the midgut. We predicted that anti-AgGluCl IgG's mode of action is by antagonizing GluCl activity. To test this hypothesis, we fed A. gambiae blood meals containing anti-AgGluCl IgG and the GluCl agonist ivermectin (IVM). Anti-AgGluCl IgG attenuated the mosquitocidal effects of IVM, suggesting that anti-AgGluCl IgG antagonizes IVM-induced activation of GluCl. Lastly, we stained adult, female A. aegypti and C. tarsalis for GluCl expression. Neuronal GluCl expression in these mosquitoes was similar to previously reported A. gambiae GluCl expression; however, we also discovered GluCl staining on the basolateral surface of their midgut epithelial cells, suggesting important physiological differences in Culicine and Anopheline mosquitoes.

Sampling host-seeking anthropophilic mosquito vectors in west Africa: comparisons of an active human-baited tent-trap against gold standard methods.

In this study, we characterize the ability of the previously described Infoscitex tent (IST) to capture mosquitoes in comparison to either the Centers for Disease Control Light Trap hung next to individuals under a bed net (LTC) or to human landing catches (HLC). In Senegal, the IST caught 6.14 times the number of Anopheles gambiae sensu lato (s.l.), and 8.78 times the Culex group V mosquitoes as LTC. In one of two locations in Burkina Faso, the IST caught An. gambiae at a rate not significantly different than HLC. Of importance, 9.1-36.1% of HLC caught An. gambiae were blood fed, mostly with fresh blood, suggesting they fed upon the collector, whereas only 0.5-5.0% from the IST had partial or old blood. The IST also caught outdoor biting species in proportions comparable to HLC. The results show this tent provides a safer and effective alternative to the skill-dependent, risky, and laborious HLC method.

Evaluation of ivermectin mass drug administration for malaria transmission control across different West African environments.

BACKGROUND: Mass drug administration (MDA) of ivermectin to humans for control and elimination of filarial parasites can kill biting malaria vectors and lead to Plasmodium transmission reduction. This study examines the degree and duration of mosquitocidal effects resulting from single MDAs conducted in three different West African countries, and the subsequent reductions in parity and Plasmodium sporozoite rates. METHODS: Indoor-resting, blood-fed and outdoor host-seeking Anopheles spp. were captured on days surrounding MDAs from 2008-2013 in Senegalese, Liberian and Burkinabé villages. Mortality was assessed on a portion of the indoor collection, and parity status was determined on host-seeking mosquitoes. The effect of MDA was then analysed against the time relative to the MDA, the distributed drugs and environmental variables. RESULTS: Anopheles gambiae survivorship was reduced by 33.9% for one week following MDA and parity rates were significantly reduced for more than two weeks after the MDAs. Sporozoite rates were significantly reduced by >77% for two weeks following the MDAs in treatment villages despite occurring in the middle of intense transmission seasons. These observed effects were consistent across three different West African transmission dynamics. CONCLUSIONS: These data provide a comprehensive and crucial evidence base for the significant reduction in malaria transmission following single ivermectin MDAs across diverse field sites. Despite the limited duration of transmission reduction, these results support the hypothesis that repeated MDAs with optimal timing could help sustainably control malaria as well as filarial transmission.

Experimental approaches to microarray analysis of tumor samples.

Comprehensive measurement of gene expression using high-density nucleic acid arrays (i.e. microarrays) has become an important tool for investigating the molecular differences in clinical and research samples. Consequently, inclusion of discussion in biochemistry, molecular biology, or other appropriate courses of microarray technologies has become essential in training the modern scientist. The following article offers preparatory and problem-solving questions for engaging students in the understanding of microarray technologies for use in conjunction with other course materials relating to discussions of microarray technology.