Mosquito Host-Seeking Diel Rhythm and Chemosensory Gene Expression is Affected by Age and Plasmodium Stages

doi:10.21203/rs.3.rs-1114857/v1

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Mosquito Host-Seeking Diel Rhythm and Chemosensory Gene Expression is Affected by Age and Plasmodium Stages

https://doi.org/10.21203/rs.3.rs-1114857/v1

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Malaria parasites can affect vector-related behaviours, increasing transmission success. Using Anopheles gambiae and Plasmodium falciparum, we consider the effect of interaction between infection stage and vector age on diel locomotion in response to human odour and the expression of antennal chemosensory genes. We demonstrate age-dependent behavioural diel compartmentalisation by uninfected females. Infection disrupts overall and diel activity patterns compared with age-matched controls. Mosquitoes carrying transmissible sporozoites are more active, shifting activity periods to coincide with human host availability, in response to human odour. Older, uninfected females reduce activity during their peak host-seeking period in response to human odour. Age- and infection stage-specific changes in odour-mediated locomotion coincide with altered transcript abundance of select chemosensory genes providing a possible molecular mechanism regulating the behaviour. Our results support the hypothesis that vector-related behaviours of female mosquitoes are altered by infection stage and further modulated by the age of the vector, and have important implications for malaria transmission and disease dynamics.

Molecular Biology

Physiology

Behavioral Ecology

Plasmodium falciparum

parasite stages

Anopheles gambiae

vector

locomotion

olfaction

antennae

transcriptomics

The presence of different life-cycle stages of the malaria parasite, Plasmodium falciparum, together with the age of the vector, affect the host-seeking daily rhythm and antennal chemosensory gene expression of Anopheles gambiae to indirectly increase the odds of successful disease transmission.

Malaria parasites depend on mosquito vectors for transmission from one host to another. Increasing empirical evidence demonstrates that insect-borne pathogens may manipulate both their human host and their insect vector to enhance transmission success^1–4. This manipulation is not static, as vector-related behaviours can vary significantly between uninfected and infected vectors over different time scales relevant to the disease transmission dynamics^3–5. Pathogens are known to affect vector behaviours, including locomotion, flight, host seeking, probing and feeding, which can manifest in increased avidity to locate and obtain a blood meal or in alterations in host preference, resulting in an enhanced transmission through the increased contact between vector and host^3,5,6. The molecular mechanisms by which these behavioural traits may be modulated have only recently received attention, identifying pathways in the neuroendocrine circuitry as key targets^2,5,7,8.

Mosquitoes acquire Plasmodium parasites, the causative agent of malaria, through a blood meal from an infected vertebrate host. The pathogen undergoes several developmental stages in the mosquito, from midgut invasion and oocyst formation (infected mosquitoes, 7 days post-infection; dpi) through to the invasion of the salivary glands, when a mosquito can transmit the malaria sporozoites (infectious mosquitoes, 14 dpi)⁹. The avidity of mosquitoes to engage in vector-related behaviours, including host seeking and blood feeding, depends on the developmental stage of the pathogen in the mosquito, rather than the infection status of the mosquito alone³. Available data for Plasmodium parasites that cause avian, rodent and human malaria emphasise that vector-related behaviours are suppressed when mosquitoes are infected, but not able to transmit the pathogen, whereas infectious mosquitoes are more active^10–12. This modulation appears to be conserved across vectors and other pathogens, e.g., Aedes aegypti that transmit dengue and Zika virus^5,13. The observed changes in the odour-mediated host-seeking behaviour, demonstrated for both infected and infectious mosquitoes, are reflected in the selective sensitivity of the peripheral olfactory system to key host-related volatile organic compounds^5,10,14. Such modulation may be regulated through alterations in neural signalling and chemosensory gene expression, as demonstrated in malaria- and dengue-infected mosquitoes^2,5.

In depth analysis of the molecular basis of olfaction in mosquitoes, has allowed for the identification and functional characterisation of chemosensory gene families, including odorant receptors (Ors), gustatory receptors (Grs), ionotropic receptors (Irs), odorant binding proteins (Obps) and chemosensory proteins (Csps)^15–19. The expression of select members of these gene families in olfactory-related tissues change in response to adult maturation, age and physiological state^20–25, including infection status⁵. These state-dependent changes in gene abundance coincide with concerted alterations in the sensitivity of olfactory sensory neurones and the behaviour of the mosquitoes^20,21,25.

This study analysed the stage-dependent effect of P. falciparum infection on the locomotion activity of female Anopheles gambiae in the presence and absence of human odour, demonstrating that infected and infectious mosquitoes display differential locomotor activity correlating with age, infection status and time of day. The potential molecular mechanisms underlying the age- and infection-dependent odour-mediated locomotor activity were assessed by transcriptome analysis, identifying differentially abundant chemosensory-related genes in the antennae coinciding with the demonstrated differences in mosquito behaviour. A better understanding of the effect of infection on locomotor activity and odour-mediated host seeking throughout the day has the potential to more accurately direct vector measures. Identifying the molecular mechanisms by which these behavioural traits are modulated by P. falciparum may lead to a better understanding of vectorial capacity and transmission dynamics potentially inspiring the development of innovative control strategies.

Mosquito age and the stage of Plasmodium falciparum infection affect locomotor activity

Locomotor activity of uninfected female An. gambiae in the absence of human odour significantly reduced with age when assessed in Drosophila activity monitors (χ²₁ = 5.86, p = 0.02; Fig. 1a-b). This age effect was not observed following infection with P. falciparum (χ²₁ = 0.83 p = 0.35; Fig. 1b). In the presence of human odour, the age-dependent reduction in locomotor activity was observed in both uninfected (χ²₁ = 21.12, p <0.001) and P. falciparum carrying mosquitoes (χ²₁ = 5.73, p = 0.02; the GLMM:lmer model was constrained by age and the random effect of experimental replication; Fig. 1b).

Infection status significantly affected the activity of mosquitoes when comparing the overall locomotion profiles of 7 dpi and 14 dpi individuals with age-matched uninfected females in the presence and absence of human odour (β-estimated = weighted mean value based on all variables plus the random effect of the replication ± SE; presence of human odour: χ²₁ = 18.06, p < 0.001; absence of human odour: χ²₁ = 5.73, p < 0.01). In the absence of human odour, 7 dpi females were less active compared to age-matched controls (χ²₁ = 8.21, p = 0.004), whereas 14 dpi females were as active as their age-matched controls (χ²₁ = 2.27, p = 0.13; Fig. 1c). The presence of human odour differentially altered the locomotor activity. While there were no significant differences between 7 dpi and age-matched females in the presence of human odour (χ²₁ = 0.12, p =0.73), the infectious 14 dpi mosquitoes were more active than age-matched uninfected females (14 days control; 14 dc χ²₁ = 8.80, p = 0.002; Fig. 1c).

Mosquito locomotor activity varies temporally in response to host odour and P. falciparum infection

Diurnal cycles in An. gambiae locomotor and host-seeking activities were evident, with distinct crepuscular activity peaks culminating in the highest activity at the onset of photophase (ZT 23-24), and low to moderate activity during scotophase (ZT 12-23) and low activity during photophase (ZT 0-11)²⁶. To assess the diurnal effect on the locomotor activity of mosquitoes with P. falciparum infection and their respective controls, a mixed model analysis was used (lmer model: constrained by two main explanatory variables, infection status and period of time, as well as the random effect of replication, Supplementary Table 1-2). In the absence of human odour, the diurnal locomotor activity profile of uninfected mosquitoes confirmed the high activity at dawn (ZT 23-24), with the younger cohort being more active during scotophase with a peak at dawn, whereas the older control females displayed an increased activity at dusk that was maintained until dawn (ZT 0-11: χ²₁ = 27.43, p < 0.001; ZT 11-12: χ²₁ = 4.58, p = 0.049; ZT 12-23: χ²₁ = 4.33, p = 0.03; ZT 23-24: χ²₁ = 32.32, p < 0.001, Fig. 1d, Supplementary Table 2.). In the presence of human odour, the locomotor activity of the younger uninfected cohort increased at dawn, and remained at this activity level until dusk, while the older females only displayed an increase in activity at dusk (ZT 0-11: χ²₁ = 17.54, p < 0.001; ZT 11-12: χ²₁ = 7.67, p = 0.005; ZT 12-23: χ²₁ = 15.21, p < 0.001; ZT 23-24: χ²₁ = 18.25, p < 0.001, Fig. 1d, Supplementary Table 2.).

The time-of-day, and the stage of P. falciparum sporogony, differentially affected the locomotor activity of the vector (Fig. 1d). In mosquitoes with parasites undergoing sporogony, there was no effect of age on locomotion throughout the 24 h period in the absence of human odour (ZT 11-12: χ²₁ = 1.71, p = 0.18; ZT 12-23: χ²₁ = 0.31, p = 0.57; ZT 23-24: χ²₁ = 3.49, p = 0.06, Supplementary Table 2.), except during photophase, in which infectious (14 dpi) mosquitoes demonstrated lower activity than infected (7 dpi) individuals (ZT 0-11: χ²₁ = 21.97, p < 0.001). However, in the presence of human odour, the infectious cohort (14 dpi) was more active at dawn (ZT 23-24: χ²₁ = 15.40, p < 0.001), while being less active than the younger mosquitoes throughout the rest of the diurnal period (ZT 0-11: χ²₁ = 9.35, p = 0.002; ZT 11-12: χ²₁ = 20.02, p < 0.001; ZT 12-23: χ²₁ = 9.73, p = 0.001, Supplementary Table 2.).

Individual locomotor profiles of mosquitoes at 7 dpi and 14 dpi differed significantly from that of age-matched uninfected controls (Fig. 1d). Infection significantly reduced the locomotor activity in 7 dpi females in the absence of human odour when compared with their uninfected counterparts during most time periods (ZT 0-11: χ²₁ = 8.49, p = 0.03; ZT 11-12: χ²₁ = 1.008, p = 0.31; ZT 12-23: χ²₁ = 7.84, p = 0.005; ZT 23-24: χ²₁ = 13.78, p < 0.001, Supplementary Table 1-2). Mosquitoes carrying the transmissible stage of the parasite (14 dpi) displayed a lower locomotor activity than the uninfected counterparts throughout photophase and scotophase (ZT 0-11: χ²₁ = 5.92, p = 0.01; ZT 12-23: χ²₁ = 11.87, p < 0.001, Supplementary Table 1-2), but not at dusk and dawn (ZT 11-12: χ²₁ = 2.68, p = 0.10; ; ZT 23-24: χ²₁ = 2.20, p = 0.10). In the presence of human odour, the infected (7 dpi) females showed lower activity than age-matched controls at dawn (ZT 23-24: χ²₁ = 7.29, p = 0.006), while the inverse activity pattern was observed during photophase (ZT 0-11: χ²₁ = 12.28, p = 0.001). The infectious mosquitoes (14 dpi) demonstrated a significantly increased locomotor activity at dawn (ZT 23-24: χ²₁ = 23.78, p < 0.001) and during scotophase (ZT 12-23: χ²₁ = 25.42, p = 0.001), while there was no difference in activity of the infectious and uninfected females during photophase (ZT 0-11: χ²₁ = 2.26, p = 0.13), with a significant reduction in dusk (ZT 11-12: χ²₁ = 16.06, p < 0.001, Supplementary Table 1- 2).

Plasmodium falciparum modulates antennal transcript abundance

Paired-end sequencing of each of the libraries constructed from antennal RNA, with a total of 2 400 antennae, generated an average mapping of 26 399 740 million cleaned reads per library. Out of the 13 832 coding genes annotated in the genome of An. gambiae (Agam4.10), a total of 10 115 transcripts were reliably detected above 1 transcript per million (TPM) mapped reads in the antennae, among all experimental and control groups, demonstrating an adequate level of coverage.

A principal component analysis (PCA) of the antennal transcripts was conducted to demonstrate the overall variation among the antennal transcriptomes (infected, infectious, and age-matched uninfected conditions; 4 replicates each; Fig. 2a). The PCA identified that 49.8% of the variation among the libraries was based on the relative infection status in each age group (PC1), while 15.3% of the variance was dependent on age and infection status relative to the controls (PC2; Fig. 2a). All of the biological replicates of the same age and infection status clustered tightly together in the principal component space, except for the infectious samples (14 dpi), demonstrating that variation in the libraries due to handling and processing was successfully minimised. The separation of the four libraries of the 14 dpi samples into two clusters correlates with demonstrated differences in parasite load of the mosquitoes (Fig. 2a; Supplementary Fig. 1).

Antennal transcripts were significantly differentially regulated between the two age-matched cohorts (6 187), of which 3 465 were differentially abundant only between 14 dpi and 14 dc, whereas in 7 dpi and 7 dc, 850 were differentially abundant (Fig. 2b). In total, 4 807 transcripts were significantly differentially regulated between the two age-matched control groups and the two groups carrying P. falciparum parasites, of which 1 071 transcripts were shared between them (Fig. 2c). Within the age-matched control groups, 2 614 transcripts were uniquely regulated, whereas 1 122 were uniquely regulated within the two groups carrying P. falciparum parasites (Fig. 2c; Supplementary Fig. 2). Overall, differentially abundant transcripts were not condition-dependently regulated, except those regulated post-infection during both the infected and infectious stages (1 872), of which more than 80% of the transcripts were down-regulated post-infection (Fig. 2b).

To characterise the functional ontology of the differentially abundant genes in the antennae of infected and infectious mosquitoes with their age-matched controls, a gene ontology (GO) analysis of molecular function (level three) was conducted (Fig. 3). Of the 3 685 differentially abundant transcripts between the two uninfected control groups, the majority (>75%) were functionally classified as structural constituent of cuticle (GO: 0042302) and enzyme inhibitor activity (GO: 0004857; Fig. 3a). Both of these classes were more abundant in older compared to the younger individuals. None of the age-dependent GO terms identified in the uninfected cohort comparison (Fig. 3a) were detected in the pairwise comparisons of the antennal transcriptomes among infected, infectious and their age-matched controls. The three most represented functional classes in the pairwise comparisons between the infected and infectious groups with their age-matched controls, as well as between the infected and infectious antennal transcriptomes, were heterocyclic compound binding (GO: 1901363), organic cyclic compound binding (GO: 0097159) and ion binding (GO: 0043167; Fig. 3b). Two functional classes were regulated differently in the antenna of mosquitoes with a P. falciparum infection, odorant binding (GO: 0005549) and carbohydrate derivative binding (GO:0097367). The number of genes in the functional class odorant binding were regulated in both infected and infectious samples, while those in the carbohydrate derivative binding class were differentially regulated only in infectious samples (Fig. 3b).

A detailed analysis of the major chemosensory gene families associated with the odorant binding functional class, Ors, Irs, Grs, Csps and Obps, was conducted. As the mosquitoes aged, 34% of the chemosensory genes were significantly regulated in the antennae of uninfected females, with chemosensory receptors demonstrating higher abundance in the older females, while the binding proteins were both up- and down-regulated (Fig. 4). Following P. falciparum infection, the abundance of 18 Obps reduced with age, while only one chemosensory gene, Ir41a, demonstrated an increased abundance in the antennae of older females (Fig. 4). The differential abundance of these 18 Obps, along with 3 others, appear to be a result of an increased abundance at 7 dpi compared to the age-matched controls. The other chemosensory gene that was regulated at this age, Ir7u, was down-regulated upon infection.

The age-dependent regulation of chemosensory genes was affected following P. falciparum infection, as 73% of those genes that were up-regulated with age (Fig. 4 column 1), were shown not to increase in abundance post-infection (Fig. 4 columns 2 and 3), resulting in a higher abundance of these transcripts in the controls compared with those at 14 dpi (Fig. 4 column 4). The only exception to this was Ir7u, which was down-regulated at 7 dpi, exacerbating the decreased abundance observed at 14 dpi. Interestingly, when the 7 dpi mosquitoes were compared to 14 dpi, the abundance of the above-mentioned Obps were reversed, returning to pre-infection levels (Fig. 4 column 2).

The stage of infection of P. falciparum in An. gambiae has a significant impact on vector-related behaviour, which affects malaria transmission and disease dynamics^2,5,27,28. This study confirms that infected mosquitoes are less active than infectious females, and furthermore identifies this change in behaviour to be a result of an interaction between infection stage and age, which can be modulated by the presence of human odour and diel rhythms. Transcriptomic profiling of antennal tissue of P. falciparum-infected and -infectious An. gambiae identifies members of several chemosensory gene families as changing with age and infection status, suggesting a molecular mechanism, which may regulate the sensitivity of the peripheral olfactory system of the vector to human odour during the sporogonic cycle. As age is intrinsic to the infection status of the female, controlling for age provides a more accurate indication of which behaviours and molecular correlates are regulated by P. falciparum infection.

Host seeking in mosquitoes is dependent on age^{20,21,23,29,30} and physiological state, including infection^3–5. Our results demonstrated that the overall locomotor activity of uninfected females significantly reduced with age, in line with previous studies on mosquitoes^21,23, further emphasising the role of age in regulating host seeking. As mosquitoes age, energy reserves decrease, resulting in older females reducing their search activities and becoming more selective to reduce their energy requirements^31,32. The presence of human odour did not alter the overall locomotor activity, similar to that described by Cator et al.^10,28, suggesting that the difference in the level of activity with age is independent of the presence or absence of human odour. While previous studies on the effects of Plasmodium infection on mosquito host seeking have shown a reduced activity in infected compared with infectious mosquitoes, these have not controlled for the age of the insects^10,11. Our results indicate an interaction between age and infection in infected females that display a similar locomotor activity as an age-matched cohort, which is not seen in infectious individuals. When controlling for age and observing the effect of infection, infected females reduced their overall locomotor activity compared to age-matched controls in the absence of human odour, while in the presence of human odour there was no discernible difference in activity. In contrast, infectious mosquitoes increased their overall locomotor activity in the presence of human odour, but not in its absence, compared to age-matched controls. These results provide a rationale to explain why previous studies have presented opposing findings concerning the activity of oocyst-infected and sporozoite-infectious females^10,33. The mechanisms underlying the changes in host-seeking and blood-feeding behaviours are still unclear, however several hypotheses have been advanced, chief among these are that the response in infected and infectious females is a result of a general immune response, and/or a direct manipulation of the vector behaviour by the pathogen^3,34−36. The increased response to human odour by infectious mosquitoes coincides with increased feeding frequency, probing rate and meal size^28,35,37,38, and has substantial effects on malaria transmission ecology and overall disease dynamics³⁶.

Diel locomotor activity in mosquitoes is regulated by the age and infection status of female malaria vectors^26,39,40. In the presence and absence of human odour, younger uninfected mosquitoes (13 days post-emergence, dpe) were more active than the older mosquitoes (19 dpe) during photophase, scotophase and peaking at dawn⁴¹. The peak of activity for older females, on the other hand, occurred at dusk. The age-dependent change in diel locomotor activity was not modulated by the presence of human odour, which is in line with the diel patterns of the peripheral olfactory system responsiveness^42–44. The shift in peak activity of older females co-occurs with an increased propensity of older females to visit nectar sources at dusk⁴⁵, indicating a behavioural compartmentalisation due to an age-related reduction in energy reserves^31,46. Infection with P. falciparum altered the diel locomotor activity in both the presence and absence of human odour, generally decreasing activity in infected and infectious females in the absence of human odour, similar to that described in An. stephensi infected with Plasmodium yoelii⁴⁷. In the presence of human odour, infected and infectious females demonstrated a disrupted activity pattern associated with host seeking. In particular, infectious females shifted their odour-associated activity to scotophase and dawn, which is in line with observations of field-caught mosquitoes carrying sporozoites^39,48. By selectively increasing their activity during times of low-risk of predation and high access to human hosts, the infectious females increase the probability of a successful blood meal and pathogen transmission^49–53. While the infectivity of the bite is not affected by diel rhythms⁵⁴, the altered host-related activity pattern in infectious females may have explicit implications on the most effective deployment of vector control tools and thus our ability to affect malaria transmission dynamics efficiently³⁶. Whether this is a result of an active manipulation by the pathogen or a general immune response remains to be clarified^{3, 34–36,55,56}.

Age and P. falciparum infection significantly affected overall antennal transcript abundance, with infection systemically down-regulating gene abundance, and differentially affecting the functional ontological class odorant binding (GO:0005549). Among the differentially abundant transcripts, antennal abundance generally increased with age across all of the major olfactory-related gene families, whereas only Obps, the most abundantly expressed transcripts of the chemosensory gene families, exhibited higher abundance in infected compared with infectious females. Odorant binding proteins are involved in binding and transporting odorants to the receptors and in the overall sensitivity of the peripheral olfactory system by providing gain control^22,57,58. The Obps appear to play a major role in regulating the sensitivity and selectivity of the peripheral olfactory system of An. gambiae in response to aging and infection status, as 67% of the Obp transcripts reliably expressed in the antennae were age- and infection-dependently regulated. As the females age, older individuals exhibited a higher transcript abundance in two thirds of the differentially expressed classic, and all of the plus-C, classified Obps^17,57. The other third of the differentially regulated classic Obps, along with all 13 of the two-domain Obps^17,57, were expressed abundantly in 7 dpi antennae compared with not being reliably expressed in their age-matched controls and infectious females. Bimolecular protein interaction models indicate that the most energetically favourable ligands of the reliably expressed classic Obps, including those that were differentially abundant, are distinct sets of carboxylic acids, aromatics and terpenes¹⁷, known to regulate host and floral seeking^45,46. While no clear pattern of ligand binding was apparent associated with aging, those classic Obps increasing in abundance 7 dpi generally did not include carboxylic acids among their modelled ligands, likely resulting in a reduced sensitivity of the peripheral olfactory system to these salient human odorants by infected females^59–61. This corresponds to the reduction in human odour-associated activity exhibited by infected females.

The functionally characterised differentially abundant olfactory receptors, Or11, Or39 and Ir41a, all respond to human odour constituents^16,21,62. The receptor Or39 has been postulated to modulate the onset of host seeking in Anopheles coluzzii through the concerted downregulation of the receptor and the sensitivity to sulcatone of its cognate olfactory sensory neuron²¹. Whether the lower abundance of Or39, in conjunction with Or11, observed in the antennae of infectious compared with uninfected females underlies the observed increased locomotor activity of infectious females in the presence of human odour, remains to be investigated. The only receptor to have a higher abundance in the antennae of infectious compared to infected females, Ir41a, responds to the human spermous cyclic amine pyrrolidine^16,63, suggesting that this receptor is involved in the increased attraction to human odour 14 dpi. The differential regulation of transcript abundance of genes modulating the chemosensory pathway in response to parasite development within the insect vector provides a mechanism by which the physiological¹⁴ and behavioural sensitivity^{4,10,11,33,35} to human odour can be modulated during the sporogonic cycle of P. falciparum.

Behavioural activity of An. gambiae and its expression of chemosensory-related genes are altered in an age- and P. falciparum stage-dependent manner. Irrespective of the mechanism regulating these changes, be it active manipulation by the parasite and/or general response to immune challenge, the differential behavioural response to human odour of females as they age or become infected significantly affects vectorial capacity and malaria transmission. Our ability to increase the resolution concerning which behavioural change is affected by age and/or infection, allows us to fine tune the epidemiological models for malaria transmission³³, and integrate both factors and their interactions into the development and implementation of control measures.

Ethics

Human blood (type O) was provided in citrate-phosphate-dextrose-adenine anti-coagulant/preservative, and serum (type AB) was obtained from the blood transfusion service (blood bank) at the Karolinska University Hospital (Solna, Sweden), in accordance with the Declaration of Helsinki, and approved by the Ethical Review Board in Stockholm (2011/850-32). The human materials were obtained anonymously from the blood bank and did not require an informed consent statement. The Swedish Work Environment Authority (Stockholm, Sweden, SU FV-2.10.2-2905-13/31-01-2022) approved the class 3 biological agent laboratory and its practices, including insectary design and equipment to work with P. falciparum-infected mosquitoes. The regulations are mainly based on the EU directive 2000/54/EC on the protection of workers from risks related to exposure to biological agents at work.

Mosquito rearing and blood feeding

Anopheles gambiae (Keele) were reared and maintained at 27 ± 1 °C, 80 ± 1% relative humidity and a photoperiod of 12 h light: 12 h dark cycle, 11 h full light of ~ 300 lx and 11 h darkness, separated by 1 h dawn and dusk transitions, respectively. The Keele colony was established at Keele University as a result of balanced interbreeding of four An. gambiae s.l. strains: G3 from MacCarthy Island, the Gambia; and three Tanzanian strains, ZAN U from Zanzibar, Ifakara strain from Njage, and KIL from Marangu⁶⁴, and set up at Stockholm University in 2014. For experiments, pupae (ca. 350) were collected and transferred to cages (custom made: 30 cm × 20 cm × 20 cm) to emerge as adults, which were fed ad libitum on 5% glucose and 0.05% para-amino benzoic acid (PABA, Sigma-Aldrich, Steinheim, Germany). Females (5 dpe) were provided with a meal of cultured red blood cells (RBCs) through glass membrane-feeders connected to a custom-built heating system kept at 37 °C. The RBCs were washed with Roswell Park Memorial Institute (RPMI-1640) medium (Gibco™, ThermoFisher Scientific, Drieich, Germany), and stored in RPMI-1640 at 50% haematocrit at 4 °C prior to use.

Parasite culture and mosquito infection

The strain of P. falciparum used in this study, NF₅₄-SU, was initially isolated in the Netherlands and donated by Klavs Berzins. In vivo culturing of P. falciparum NF₅₄-SU followed the standard protocol⁶⁵. In short, P. falciparum NF₅₄-SU was cultured using washed RBCs (5% haematocrit) from type-O blood donors, and 10% serum mixed with RPMI-1640 medium (Gibco™, ThermoFisher Scientific), supplemented with 5.94 g l^-1 HEPES buffer (Sigma Aldrich), 2.1 g l^-1sodium hydrogen carbonate (Sigma Aldrich), and 0.05 g l^-1 hypoxanthine (Sigma Aldrich). The cultures were kept in controlled conditions under gas (1% O₂, 3% CO₂ and 96% N₂) at 37 °C. Plasmodium falciparum gametocyte cultures were set up at 0.5-0.7% parasitaemia, 6% haematocrit in RPMI-1640 medium. On the day of experimental infection, uninfected RBCs were supplemented with P. falciparum gametocytes to a final gametocytaemia of ca. 0.7%, which generates a high infection prevalence (>50%) in An. gambiae s.s⁶⁵. For each infectious meal, mixtures of gametocytes from 14- and 17-day gametocyte cultures were used. Twenty-eight groups of ca. 100 mosquitoes were fed on mature gametocyte-infected RBCs or on uninfected washed RBCs using a membrane feeder. All age-matched control females ( 7 and 14 days control ; 7 and 14 dc) were handled in the same manner as the infected and infectious cohorts, including access to mates, blood meals and oviposition substrates.

Mosquito odour-mediated locomotor activity

For recording the behavioural activity of the mosquitoes, DAM2 Drosophila activity monitors (TriKinetics Inc., Waltham, MA, USA) were used. Two hours prior to the experiments, mosquitoes were placed individually in glass tubes (65 mm ⅹ 7 mm), using an aspirator, for pre-conditioning. Cotton plugs saturated with 5% glucose with 0.05% PABA (as above) were placed at one end of the glass tubes to provide ad libitum access to sustenance. Human odour was supplied by a previously worn cotton sock (ca. 24 h) placed inside a 200 ml glass wash bottle (Sigma Aldrich). The odour was introduced, via Teflon tubing, into the glass tubes through a MAN2 gas distribution manifold (TriKinetics Inc.). A clean unworn sock was similarly used as a control. Each experiment was replicated three times with infected and infectious mosquitoes assessed in parallel with age-matched controls, for a total of 384 mosquitoes assayed (96 per condition). Behavioural experiments were conducted in the class 3 biological agent laboratory (BSL3) within a climate chamber kept at 27 ± 1 °C, 70% relative humidity with a photoperiod of 11 h light: 11 h dark with 60 min of both dawn and dusk lighting conditions. Both assays were conducted over 24 h, with activity data recorded every 30 s.

The influence of age, the presence and absence of human odour, and the effect of the sporogonic stages of P. falciparum on the An. gambiae ( 7 and 14 days post infection; 7 and 14 dpi), diel locomotor activity was analysed using general linear mixed models (GLMM), in which the effect of replication (experimental blocks) and the weighting of multiple replications (random variables) were taken into account. For all results, the significance of the best maximal model containing all explanatory effects was evaluated by using likelihood ratio test. All analyses were performed using R (R Core team⁶⁶: R v.3.2.3 and RStudio 1.1.463⁶⁷). The b-estimated values of locomotor activity were estimated in all final significant models (GLMM: lmer function; in Fig 1b: The mixed model was build based on the effect of age as a main fixed variable combined with the random effect of the other variables; Fig 1c: The model was demonstrated the effect of the main fixed variable of infection including the rest of the variables as random variables; Fig 1d: The mixed model was estimated the effect of all four main fixed variables including age, infection, time, human odour, as well as the random effect of the experimental replication combined with the interactions among all these variables). In all analyses, all of the data met the assumptions of the test for normality and error homogeneity. Backward elimination was used for sequential removal of non-significant variables, to obtain the minimal statistically significant model. All graphical visualisations and statistical analyses were preformed using GraphPad Prism⁶⁸ and R (R Core team⁶⁶: R v.3.2.3 and RStudio 1.1.463⁶⁷). The precise levels of significance are presented in the results section, except the universally accepted p < 0.001.

Transcriptome analyses

Pairs of antennae from infected, infectious and age-matched control mosquitoes were collected from individual females following flash freezing on dry ice for <30 s, and placed into 24-well culture plates (ThermoFisher Scientific) in RNAlater (500 µl; Thermo Fisher Scientific). Individual carcasses (bodies missing antennae) of infected and infectious mosquitoes were also collected for subsequent qPCR analysis, to determine the parasite abundance of individual females (see below). Following the quantification of the parasite load, the antennae of successfully infected individuals were pooled into tubes of 150 pairs of antennae, according to the level of infection, and stored at -80 °C. Four replicates of each condition were collected. RNA libraries were constructed using a TruSeq RNA Library Prep Kit (Illumina, Berlin, Germany) from total RNA from the pooled antennae following extraction and DNase I digestion (RNeasy Mini kit, Qiagen, Hilden, Germany). Quality and quantity control of total RNA aliquots was performed using a Nanodrop spectrophotometer (Thermo Fisher Scientific), an Agilent 2100 Bio-analyser (Santa Clara, CA, USA) and a Qubit 2.0 Fluorometer (Thermo Fisher Scientific), before sending to BGI Genomics (MGI Tech Co., Ltd., China) for Illumina paired-end indexed sequencing, using the Illumina HiSeq2000.

RNA-Seq and differential expression analyses

High-quality reads were determined using the following criteria: low quality reads were clipped from the start and end of each read using a sliding window and reads shorter than 40 nt were removed. The cleaned reads were mapped using CLC Genomics Workbench version 11 (https://digitalinsights.qiagen.com) to the An. gambiae genome Agam4 with reference to the Agam4.10 gene set (www.vectorbase.org). On average 94.11% of all reads mapped to the genome. Transcript abundance was reported as transcript per million (TPM) with a threshold level of abundance above 1 TPM. Differential transcript abundance was determined using the β-binomial general linear model algorithms in CLC Genomics Workbench 11. To control for false discovery rate (FDR), the Benjamini-Hochberg correction was applied⁶⁹. This analysis generated weighted fold changes (FC) and FDR-corrected p-values that were used to detect differential expression. Significantly differential gene abundance was determined based on a FC ≥ 2 and a FDR-corrected p-value < 0.05.

Quantitative real-time polymerase chain reaction analysis

Quantitative real-time polymerase chain reaction(qPCR) was used to estimate the abundance of P. falciparum in individual mosquitoes subjected to an infectious meal at 7 and 14 dpi, and used for both behavioural and transcriptomic analyses, allowing quantification of the numbers of parasite genomes. In all analyses, a single body of an uninfected female mosquito was used as a negative control. Reactions were performed on a Roche Light Cycler using SYBR Green (Roche, Mannheim, Germany). DNA standards containing known numbers of P. falciparum parasites were produced from asexual cultures, and used to generate standard curves of P. falciparum. A similar statistical approach, as described for the behavioural analyses, was used to test for variation in the number of parasites (oocyst load and total number of sporozoites per whole mosquito body)⁷⁰. Given the highly over-dispersed nature of parasite abundance data, a negative binomial distribution was assumed in these analyses (GLMM: glmmADMB, nlme package; R Core team⁶⁶: R v.3.2.3 and RStudio 1.1.463⁶⁶). A backwards elimination approach was used to test for the significance of all fixed effects and interactions, while controlling for random variation due to replicate, as for the analyses of oocyst and sporozoite prevalence.

Data availability

All data supporting the findings of this study are available within the article and its Supplementary Information files. The sequencing data are available at the GEO database (BioProject ID PRJNA756244), and are available from the corresponding authors upon request.

Acknowledgements

We thank the Swedish Research Council for funding to RI (VR/2014-3331) and SNE (VR/2017-01229; VR/2017-05543 UFNW). We extend our thanks to Jeansson´s Stiftelser (SJ-2018) for the valuable support for setting up the essential equipment of the BSL3 laboratory. We highly appreciate the donation of the NF₅₄-SU strain of P. falciparum by Prof. Klavs Berzins.

Author Contributions

RI and SNE conceived the study and designed the experiments. SNE and MH cultured the parasites, performed the mosquito infections as well as conducting the behavioural analyses. All statistical and modelling analyses for behavioural assays were carried out by MH and SNE for the deep analysing of the mixed model data. LRC, RM and SNE dissected the antenna and prepared RNA for Illumina sequencing. RI, SRH and SNE designed the transcriptome study, and MH and SRH performed the analyses in consultation SNE. MH, RI, SRH and SNE wrote the manuscript. All authors read and approved the manuscript.

Competing interests

The authors declare no competing interests. S. Noushin Emami is a co-founder of Molecular Attraction AB and there are no commercial aspects or financial gain to this company resulting from this study.

Materials and correspondence

Correspondence and materials requests should be directed to S. Noushin Emami, [email protected]; and Rickard Ignell, [email protected]

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No competing interests reported.

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Mosquito Host-Seeking Diel Rhythm and Chemosensory Gene Expression is Affected by Age and Plasmodium Stages

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