Histamine Ingestion by Anopheles Stephensi Alters Important Vector Transmission Behaviors and Infection Success with Diverse Plasmodium Species

biomolecules

Article Histamine Ingestion by Anopheles stephensi Alters Important Vector Transmission Behaviors and Infection Success with Diverse Plasmodium Species

Anna M. Rodriguez 1, Malayna G. Hambly 1, Sandeep Jandu 2 , Raquel Simão-Gurge 1, Casey Lowder 1, Edwin E. Lewis 1, Jeffrey A. Riffell 2 and Shirley Luckhart 1,3,*

1 Department of Entomology, Plant Pathology and Nematology, University of Idaho, Moscow, ID 83843-3051, USA; [email protected] (A.M.R.); [email protected] (M.G.H.); [email protected] (R.S.-G.); [email protected] (C.L.); [email protected] (E.E.L.) 2 Department of Biology, University of Washington, Seattle, WA 98195-1800, USA; [email protected] (S.J.); [email protected] (J.A.R.) 3 Department of Biological Sciences, University of Idaho, Moscow, ID 83843-3051, USA * Correspondence: [email protected]; Tel.: +208-885-1698

Abstract: An estimated 229 million people worldwide were impacted by malaria in 2019. The vectors of malaria parasites (Plasmodium spp.) are Anopheles mosquitoes, making their behavior, infection

success, and ultimately transmission of great importance. Individuals with severe malaria can exhibit signiﬁcantly increased blood concentrations of histamine, an allergic mediator in humans

Citation: Rodriguez, A.M.; Hambly, and an important insect neuromodulator, potentially delivered to mosquitoes during blood-feeding. M.G.; Jandu, S.; Simão-Gurge, R.; To determine whether ingested histamine could alter Anopheles stephensi biology, we provisioned Lowder, C.; Lewis, E.E.; Riffell, J.A.; histamine at normal blood levels and at levels consistent with severe malaria and monitored blood- Luckhart, S. Histamine Ingestion by feeding behavior, flight activity, antennal and retinal responses to host stimuli and lifespan of adult Anopheles stephensi Alters Important female Anopheles stephensi. To determine the effects of ingested histamine on parasite infection success, Vector Transmission Behaviors and we quantified midgut oocysts and salivary gland sporozoites in mosquitoes infected with Plasmodium Infection Success with Diverse yoelii and Plasmodium falciparum. Our data show that provisioning An. stephensi with histamine at Plasmodium Species. Biomolecules 2021, levels consistent with severe malaria can enhance mosquito behaviors and parasite infection success 11, 719. https://doi.org/10.3390/ in a manner that would be expected to amplify parasite transmission to and from human hosts. Such biom11050719 knowledge could be used to connect clinical interventions by reducing elevated histamine to mitigate human disease pathology with the delivery of novel lures for improved malaria control. Academic Editors: Emanuela Masini and Laura Lucarini Keywords: histamine; Anopheles stephensi; flight behavior; blood-feeding behavior; infection success;

Received: 3 April 2021 lifespan; Plasmodium yoelii; Plasmodium falciparum Accepted: 5 May 2021 Published: 11 May 2021

Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in In 2019, there were an estimated 229 million cases of malaria worldwide [1]. Anopheles published maps and institutional affil- mosquitoes are the vectors of malaria parasites (Plasmodium spp.), making their behavior, iations. infection success, and ultimately their role in parasite transmission of great importance. Both children and adults with severe falciparum malaria (SFM) can exhibit significantly increased levels of blood histamine [2,3], which is generated from the amino acid histidine by the catalytic action of histidine decarboxylase (HDC), which is expressed primarily by Copyright: © 2021 by the authors. basophils and mast cells. Strikingly, blood histamine in pediatric SFM can rise to nearly Licensee MDPI, Basel, Switzerland. 10 nM or up to 480% of uninfected levels [2,3]. Increased levels of this allergic mediator in This article is an open access article blood are notable in that histamine is a significant contributor to inflammation and disease distributed under the terms and pathology not only in malaria [2,3], but also in many other disease states [4]. conditions of the Creative Commons In addition to its role in malaria pathology, histamine is an important insect neuromod- Attribution (CC BY) license (https:// ulator, highlighting the potential that this biogenic amine is ingested at bioactive levels by creativecommons.org/licenses/by/ mosquitoes feeding on gametocytemic individuals. In anopheline mosquitoes, transcripts 4.0/).

Biomolecules 2021, 11, 719. https://doi.org/10.3390/biom11050719 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 719 2 of 23

for histamine receptors and HDC are expressed in the midgut and head [5,6]. Across insect species, strong abdominal histamine receptor expression and histaminergic neurons project to the brain, where they innervate sensory brain regions to influence behavior [7–9]. In a wide variety of arthropods, signaling between the gut-brain and abdomen-brain has been shown to facilitate the processing of food and biologically important sensory (visual and olfactory) information [7–9]. Histaminergic signaling between thoracic and brain regions has also been shown to be involved in sensory modulation and flight [8,10,11]. The anopheline mosquito represents a unique situation to explore gut-brain axis biology in that these mosquitoes would be predicted to ingest varying levels of histamine at blood-feeding, bringing these compounds into direct contact with mosquito tissues, including the midgut, that express receptors and biosynthetic enzymes for biogenic amines. While it is true that increased gametocytogenesis has been correlated with SFM, specifically with patterns of increased host immunity, severe anemia, and induced reticulocyto- sis [12–16], the presence of gametocytes does not always predict successful transmission to mosquito vectors. Further, while malarial gametocytemia has been positively correlated with mosquito infectivity [17–19], gametocyte commitment can occur without the maturity to infectious stage V gametocytes, and gametocytemia is not the only predictor of mosquito infectivity [20–23]. In this context, we sought to test the hypothesis that malaria-associated changes to circulating histamine in host blood and its effects on mosquito biology could facilitate successful infection of anopheline mosquitoes by gametocytemic blood. In these studies, we provisioned histamine at normal blood levels (1 nM) and levels consistent with severe malaria (10 nM) and monitored blood-feeding behavior, flight activity, antennal, and retinal responses to host stimuli, and lifespan of adult female Anopheles stephensi, a globally important and highly invasive malaria mosquito vector that has become established in Africa [24–27]. We also evaluated the impacts of ingested histamine on An. stephensi infection with the mouse parasite Plasmodium yoelii yoelii 17XNL and the human parasite Plasmodium falciparum. Our data show that provisioning An. stephensi with histamine at levels consistent with severe malaria can enhance parasite infection and mosquito behaviors that would be expected to amplify parasite transmission to and from human hosts. Such knowledge could be used to connect clinical interventions (e.g., reducing elevated histamine to mitigate human disease pathology [28]) with the delivery of novel lures to manipulate histamine signaling in vector mosquitoes for improved future malaria control.

2. Materials and Methods 2.1. Materials Histamine (2-(1H-imidazol-5-yl)ethanamine; dihydrochloride, ACROS Organics, Fair Lawn, NJ, USA), human red blood cells (RBCs, Interstate Blood Bank, Memphis, TN, USA), and serum (Interstate Blood Bank, Memphis, TN, USA), RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with HEPES, L-glutamine, hypoxanthine (Acros Organics), DL-lactic acid (Acros Organics), Hoechst 33342-trihydrochloride trihydrate (Invitrogen, Carlsbad, CA, USA), JC-1 (Invitrogen, Carlsbad, CA, USA), mercurochrome, Ringer’s solution (3 mM CaCl2, 182 mM KCl, 46 mM NaCl, 10 mM Tris pH 7.2), 1-octen- 3-ol (Sigma-Aldrich), and electrode gel (Parker Laboratories, Fairﬁeld, NJ, USA). N-3- dimethylaminopropyl-N0-ethylcarbodiimide (Sigma-Aldrich, St. Louis, MO, USA, cat. #03449), paraformaldehyde (Electron Microscopy Sciences, Hatﬁeld, PA, USA, cat. #15710), agarose (Sigma-Aldrich), goat serum (BSA; Jackson ImmunoResearch Laboratories, West Grove, PA, USA, cat. #001-000-162), rabbit anti-histamine (ImmunoStar, Hudson, WI, USA, cat. # 22939, RRID:AB_572245), Alexa Fluor 488 (Thermo Fisher, cat. #A-11008), and Vectashield®PLUS (Vector Laboratories, Burlingame, CA, cat. #H-1900).

2.2. Mosquito Rearing Anopheles stephensi Liston, an Indian wild-type strain, were derived from the colony maintained in the Luckhart lab since 1998, at which time they were acquired from the Biomolecules 2021, 11, 719 3 of 23

long-term colony maintained in the Department of Entomology at Walter Reed Army Institute of Research (WRAIR, Washington, DC). For these studies, mosquitoes were reared and maintained under a 12 h light-dark cycle and at 27 ◦C and 80% humidity. Unless otherwise stated, adult mosquitoes were maintained on 10% sucrose-soaked cotton balls in a 1 ft3 wire mesh cage. For egg production, the adult females were allowed to feed for 30 min on live mice sedated with ketamine (50 mg/kg) and xylazine (5 mg/kg) in sterile saline. Prior to mosquito feeding, the mice were maintained in standard cages and offered free access to food and water. After the mosquito feeding was complete, the mice were euthanized by CO2 inhalation followed by cervical dislocation. All animal procedures were conducted as approved by the Institutional Animal Care and Use Committee of the University of Idaho (protocol IACUC-2020-10, approved 30 March 2020). Mosquitoes were provided oviposition substrates 2 d following the bloodmeal. Collected eggs were washed into 5 L Nalgene pans with shallow water and maintained as larvae in the same pans, on a solution of 2% powdered ﬁsh food (Sera Micron) and baker’s yeast in a 2:1 ratio for 3 d and supplemented with Game Fish Chow pellet food (Purina) until pupation. Experimental mosquitoes were 4–6 d old at the beginning of all experiments, collected at least 24 h prior to the beginning of an experiment and housed in a 500 mL polypropylene Nalgene container with mesh screening.

2.3. Techniques 2.3.1. Histamine Priming of Female An. stephensi For some studies, histamine was provisioned to mosquitoes through priming as follows. Control and treatment groups consisted of 90–120 female mosquitoes each per two- liter container provided with soaked cotton balls and a sugar cube. Controls were primed with water (control), while treatments were primed with histamine at 1 nM (healthy blood levels) or 10 nM (severe malaria [2,29]) in water via soaked cotton balls. The histamine solutions were made fresh daily, and the soaked cotton balls were changed twice daily, once in the morning between 0830–1000 and once in the evening between 1630–1800. Mosquitoes were primed for 3 d prior to the ﬁrst bloodmeal. The soaked cotton balls and sugar were removed from the cartons 30 min to 1 h prior to blood-feeding.

2.3.2. Artificial Bloodmeal Delivery to Female An. stephensi Mosquitoes were offered a bloodmeal (washed human RBCs and heat-inactivated serum 1:1 vol:vol) via an artificial feeder and were exposed to the bloodmeal for 15 min. Bloodmeals were offered in the morning between 0700–1100, following lights on at 0700. We maintained this timing for consistency throughout our studies, despite the fact that time of day for blood-feeding has been shown to minimally affect reproduction, without effects on fitness or malaria parasite infection in An. stephensi [30]. Between bloodmeals, female mosquitoes were maintained on 10% sucrose-soaked cotton balls.

2.3.3. Histamine Provisioning in a Bloodmeal to Female An. stephensi For some studies, histamine was provisioned to mosquitoes in an artiﬁcial bloodmeal as described in Section 2.3.2. Histamine was dissolved in water and diluted into the bloodmeal to ﬁnal concentrations of 1 nM or 10 nM. An identical control meal was prepared by adding a volume of water (3 µL) equivalent to that used for histamine treatments to 3 mL of the RBC-serum meal.

2.3.4. Mouse Infection with Plasmodium yoelii yoelii 17XNL Female 8–10-week-old CD1 mice were purchased from Envigo (Indianapolis, IN, USA) for parasite infection. Patterns of parasitemia and gametocytemia are indistinguishable between male and female mice, but female mice are larger and easier to manipulate, so female mice were used for these studies. Mice were maintained in standard cages and offered free access to food and water. All animal procedures were conducted as approved by the Institutional Animal Care and Use Committee of the University of Idaho (protocol Biomolecules 2021, 11, 719 4 of 23

IACUC-2020-10, approved 30 March 2020). Mice were acclimated for 1 week prior to the beginning of the study. To infect mice, 1 × 107 P. y. yoelii-infected RBCs were injected intraperitoneally into each mouse. Mice were monitored daily for parasitemia beginning at 2 d post-infection (PI) via microscopy of thin blood smears stained with Giemsa. Wet prep slides made from a drop of blood were evaluated for exflagellation of male gametocytes and counted as events per field of view prior to mosquito feeding. Mice with similar levels of exflagellation were chosen to infect mosquitoes and were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg) in sterile saline and placed on mosquito cartons for 15 min to allow mosquitoes to feed. After mosquito feeding was complete, mice were euthanized by CO2 inhalation followed by cervical dislocation per our approved IACUC protocol.

2.3.5. P. falciparum NF54 Culture Plasmodium falciparum NF54 culture was initiated at 0.5%–0.7% parasitemia. Parasites were maintained in RPMI 1640 medium supplemented with 25 mM HEPES, L-glutamine, 49 mM hypoxanthine, and 8.2 mM DL-lactic acid, with 10% heat-inactivated human serum. Parasites were synchronized by treatment with 5% sorbitol as previously described [31–33].

2.4. Impact of Histamine Provisioning on Uninfected Female An. stephensi Behavior 2.4.1. Tendency to Take a Second Bloodmeal Mosquitoes were treated with histamine by priming followed by a first bloodmeal (Section 2.3.1) or by delivery of histamine in a first bloodmeal (Section 2.3.3). After blood- feeding, unfed mosquitoes were removed and discarded. The remaining mosquitoes were provided with an oviposition substrate at 3 d post-feeding, which was removed the following day. Mosquitoes were offered a second bloodmeal at 4 d or 14 d after the first. At both 4 d and 14 d, blood-fed and non-blood-fed mosquitoes were recorded. We performed five biological replicates with both histamine priming and histamine delivery in the first bloodmeal, at each time point using separate cohorts of An. stephensi.

2.4.2. Flight Activity and Visual Object Investigation in Response to CO2 All behavioral experiments were conducted in a low-speed wind tunnel (ELD Inc., Lake City, MN, USA), with a working section 224 cm long × 61 cm wide × 61 cm high provided with a constant laminar flow of 40 cm/s. We used three short-throw projectors (LG PH450U, Englewood Cliffs, NJ, USA) and rear projection screens (SpyeDark, Spye, LLC, Minneapolis, MN, USA) to provide a low contrast checkerboard on the floor of the tunnel and grey horizons on each side of the tunnel. The intensity of ambient light from the projectors was 5 lux across the 420–670 nm range. A 3D real-time tracking system [34,35] was used to track mosquito trajectories. Sixteen cameras (Basler AC640gm, Exton, PA, USA) were mounted on top of the wind tunnel and recorded mosquito trajectories at 60 frames/s. All cameras had an opaque Infrared (IR) Optical Wratten Filter (Kodak 89B, Kodak, Rochester, NY, USA) to mitigate the effect of light in the tracking. IR backlights (HK-F3528IR30-X, LedLightsWorld, Bellevue, WA, USA) were installed below and the sides of the wind tunnel to provide constant illumination beyond the visual sensitivity of the mosquitoes. The temperature within the wind tunnel, measured using ibuttons and FLIR cameras, was a constant 22.5 ◦C and did not show any variability within the working section [34,36]. Ambient CO2 was constantly measured outside of the tunnel and was approximately 410 ppm. For each assay, 50 female An. stephensi were transferred into the tunnel and, after 1 h of acclimation, a 5% CO2 plume (or filtered air in control experiments) was released from a point source at the immediate upwind section of the tunnel at a height of 30 cm in the centerline of the tunnel. The CO2 remained on for 1 h, after which filtered air was returned to the tunnel for 1 h (post-CO2). The CO2 and filtered air were delivered using two mass flow controllers (MC-200SCCM-D, Alicat Scientific, Tucson, AZ, USA) controlled by a Python script that allowed synchronizing odor and filtered air delivery with the trajectory behaviors. To examine mosquito responses to visual stimuli, we placed 5 cm diameter Biomolecules 2021, 11, 719 5 of 23

white and black paper circles (Color-aid Corp., Hudson Falls, NY, USA) 18 cm apart on the floor of the wind tunnel in a row perpendicular to the direction of airflow. Our tracking system cannot maintain mosquito identities for extended periods of time, but individual trajectories were assumed to be independent for statistical analysis. Analyses were restricted to trajectories that were at least 90 frames (1.5 s) long. Trajectories that were at least 1.5 s were analyzed (average 3.1 s, longest 28.7 s, n = 10,635 trajectories). Flight velocities for each mosquito path were analyzed based on their 3D trajectory for the pre-CO2 (filtered air), CO2, and post-CO2 (filtered air) conditions. To examine mosquito behaviors and preferences to black and white circles in the tunnel, a fictive volume for mosquito primary activity was created around these cues (area 14 × 14 cm, height 4 cm) that was centered over the object in the crosswind direction and shifted slightly downwind in the wind line direction. The percentage of time mosquitoes investigated the objects relative to their time spent flying in the wind tunnel was also determined. In an experimental trial, approximately 10%–25% of the trajectories approached the objects. Experiments were performed with histamine-primed mosquitoes (Section 2.3.1) and mosquitoes provisioned with histamine in an RBC-serum meal (Section 2.3.3).

2.4.3. Electroretinogram (ERG) and Electroantennogram (EAG) Recordings of An. stephensi Response to Host-Associated Visual and Odor Stimuli Electroretinogram (ERG) recordings were performed by fixing individual female An. stephensi to a coverslip using Bondic glue. Mosquitoes were dark-adapted prior to stimulation. The recording glass electrode (thin-wall glass capillaries; OD, 1.0 mm; length, 76 mm; World Precision Instruments, Sarasota, FL, USA, cat. # TW100F-3) was pulled using a micropipette puller (Sutter Instrument, Novato, CA, USA, p-2000) and filled with Ringer’s solution. The reference electrode, a sharpened tungsten wire, was placed into one compound eye in a small drop of electrode gel, and the recording electrode was placed immediately on the surface of the contralateral eye. Mosquitoes were placed at the center of a semi-cylindrical visual arena (frosted mylar, 10 cm diameter, 10 cm high); a video projector (Acer K132 WXGA DLP LED Projector) was positioned in front of the arena to project the visual stimuli. To test the response to different colors, we used 2 s pulses of red, blue, or green light (distinct peaks at 460, 535, and 630 nm, 70 µW/cm2/nm), with 30 s of no light (black) between each stimulus. The colors were presented in a random order; each color was tested 10 times per mosquito, and the experiments lasted less than an hour (n = 30 mosquitoes). For the electroantennogram (EAG) recordings, the female An. stephensi head was excised, the tip of each antenna was cut off with fine scissors, and the head was then mounted on the reference electrode composed of a 0.01” silver wire (A-M Systems, Carlsbord, WA, USA). The electrode was contained in a borosilicate-pulled capillary filled with a 1:3 mix of saline and electrode gel to protect the preparation from desiccation during the experiment. The head was mounted by the neck on the reference electrode. The preparation was then moved to the recording system with the tips of the antennae inserted into a recording electrode identical to the reference electrode. The mounted antennae were oriented at 90◦ from the main airline, which was carrying filtered air (Praxair, Danbury, CT, USA). The odor stimulus was delivered using a computer-controlled solenoid system, where filtered air was passed through a 2 mL odor cartridge containing the odorant. The odorant (2 µL of 1-octen-3-ol at 10−4 dilution in mineral oil, pipetted onto a filter paper) was delivered in 1 s pulses, 10 times per mosquito (n = 15 mosquitoes). Odor and light-induced responses from the ERG and EAG recordings were amplified with an A-M Systems amplifier (10-100x; A-M Systems, 1800), digitized using a Digidata 1550B data acquisition system (Molecular Devices, San Jose, CA, USA) and analyzed using Matlab (MathWorks, Natick, MA, USA). EAG experiments used mosquitoes primed for 3 days with 10 nM histamine and control mosquitoes. For ERG experiments, we used histamine-primed mosquitoes (Section 2.3.1), and mosquitoes provisioned with histamine in an RBC-serum meal (Section 2.3.3). Biomolecules 2021, 11, 719 6 of 23

2.4.4. Histamine Immunohistochemistry Tissues (head, thorax, and abdomen) were excised and fixed in 4% N-3-dimethylamino propyl-N0-ethylcarbodiimide in 0.01 M phosphate-buffered saline (PBS) at 4 ◦C for 4 h. The tissue was then transferred and fixed in 4% paraformaldehyde in PBS overnight. Following fixation, tissues were washed in PBS and embedded in 7% agarose and sectioned at 100 µm using a Leica VT 1200S vibratome. Tissues were washed in PBS with 0.5% TritonX (PBT) and blocked in 2% goat serum for 1 h. Tissues were then incubated in 1:500 rabbit anti- histamine with 2% goat serum in PBT for 2 d at 4 ◦C. After primary antibody incubation, tissues were washed in PBT, blocked in goat serum, and incubated in 1:1000 Alexa Fluor 488. Tissues were then washed in PBT and PBS, processed through an ascending glycerol gradient and mounted in Vectashield®PLUS. The histamine antibody has shown to be specific for histamine and effective in a variety of invertebrate species, including Drosophila melanogaster [7–9]. Preabsorption controls with histamine eliminated any labeling.

2.5. Impact of Histamine Provisioning on Lifespan and Patterns of Blood Feeding of Uninfected Female An. stephensi over Time A total of 120 female mosquitoes were placed into each of three cartons. Mosquitoes in these cartons were offered a histamine-treated or control RBC-serum meal once per week as described in Section 2.3.3. Unfed mosquitoes were removed after the ﬁrst bloodmeal to ensure all mosquitoes at the beginning of the experiment imbibed the ﬁrst treatment. Control and histamine-treated bloodmeals were offered once weekly to each group until no mosquitoes remained alive. At each blood-feeding, fed and unfed mosquitoes were counted and recorded. Mosquitoes were provided with an oviposition substrate at 3 d post-feeding, which was removed the following day. Dead females were counted and removed from each group four times per week. Two biological replicates were performed with separate cohorts of An. stephensi.

2.6. Impact of Histamine Provisioning on Plasmodium Infection of An. stephensi 2.6.1. P. y. yoelii 17XNL Infection of An. stephensi and Blood Feeding Behavior of Infected Female Mosquitoes Mosquitoes were primed for 3 d (Section 2.3.1) prior to feeding on infected mice as described in Section 2.3.4. Unfed mosquitoes were removed and discarded. To quantify infection prevalence and intensity, 25–35 mosquito midguts were dissected at 10 d PI and stained with mercurochrome to count midgut oocysts. For midgut infection analysis, we performed 10 biological replicates with separate cohorts of An. stephensi. Salivary glands were dissected from 12–18 mosquitoes per group at 12–15 d PI, with infections scored on a scale of 1–4 per pair of glands, with 1 for 100–1000 sporozoites, 2 for 1000–10,000 sporozoites, 3 for 10,000–100,000 sporozoites and 4 for 100,000+ sporozoites. Salivary gland infections were analyzed from 6 of the 10 replicate infections used for midgut oocyst analysis. To examine the tendency to take a subsequent bloodmeal, two subsets of 15–20 blood- fed infected mosquitoes from each treatment group were placed in new, separate pint cartons. Mosquitoes were provided with an oviposition substrate at 3 d post-feeding, which was removed the following day, and then offered a second bloodmeal as described in Section 2.3.2 at 4 d or 11 d PI. The number of fed and unfed mosquitoes was counted and recorded. After this assay, mosquitoes were frozen and not used for other analyses. These studies were completed using mosquitoes from 5 of the 10 replicates prepared above for analyses of P. y. yoelii 17XNL midgut and salivary gland infection.

2.6.2. Impact of Histamine Treatment on P. falciparum NF54 Growth in vitro Plasmodium falciparum NF54 culture was initiated at 0.5% ring stages and maintained as described in Section 2.3.5. Treatments were prepared in duplicate, including 12 nM chloroquine as a positive control for parasite killing, along with 1, 10, and 100 nM histamine, all of which were changed daily. Plates were maintained in a candle jar [37] with Biomolecules 2021, 11, 719 7 of 23

samples collected from each well immediately after treatment (0 h) and 48 h and 96 h post treatment and stained with 12 µM Hoechst and 12 µM JC-1 to quantify parasitemia with ﬂow cytometry.

2.6.3. P. falciparum Infection of An. stephensi P. falciparum NF54 gametocyte culture was initiated at 0.7% parasitemia if synchronized rings were present or at 0.5% parasitemia if mixed stages were present. Parasites were maintained as described in Section 2.3.5. Cultures at days 14, 15, and 17 and stage IV and V gametocytes were combined and used for infections [38]. Exﬂagellation was evaluated on the day of feeding before the addition of fresh media. Immediately before feeding, histamine was added to the infected bloodmeal as described in Section 2.3.3. Mosquitoes were fed according to Section 2.3.2, after which unfed mosquitoes were removed and discarded. At 10 d PI, midguts were dissected and stained with 0.5% mercurochrome to count midgut oocysts. Four biological replicates were performed with separate cohorts of An. stephensi. Salivary glands were dissected at 15 d PI, with infections scored on a scale of 1–4 per pair of glands, with 1 for 100–1000 sporozoites, 2 for 1000–10,000 sporozoites, 3 for 10,000–100,000 sporozoites and 4 for 100,000+ sporozoites. Salivary gland infections were analyzed from 11–18 mosquitoes per treatment group from one replicate used for midgut oocyst analysis.

2.7. Statistical Analyses Data were analyzed using GraphPad (version 9.0.1, San Diego, CA, USA) or MatLab 2019a release. The numbers of mosquitoes taking a second bloodmeal and infection prevalence were analyzed using the Chi-square test. Infection intensity data were analyzed using the one-way Kruskal–Wallace ANOVA with post hoc Tukey. The EAG and ERG experiments were analyzed using an unpaired t-test or the Kruskal–Wallis test. For flight tunnel studies, mean mosquito flight velocities were calculated from the 3D tracks of each individual trajectory and data were analyzed using the Kruskal–Wallis or Mann–Whitney U-test with Bonferroni correction. Lifespan data from individual replicates were analyzed using the Kaplan–Meier analysis of survival with the log-rank test to compare groups. The proportions of mosquitoes feeding over time were analyzed using Kaplan–Meier analysis as the time to failure to feed; controls (no histamine) were not significantly different by the Wilcoxon test, so replicate data were combined for analysis. Cultured P. falciparum NF54 growth data were analyzed using ANOVA. Plasmodium falciparum salivary gland infection prevalence data were analyzed using the Chi-square test on proportional data, as several values were <5. For all analyses, significance was assumed at p ≤ 0.05.

3. Results 3.1. Histamine Provisioning of Uninfected An. stephensi Was Associated with Distinct Temporal Patterns in the Tendency to Take a Second Bloodmeal In malaria-endemic areas, the proportions of uninfected mosquitoes in natural populations are high. In sub-Saharan Africa, where the endemicity of falciparum malaria is high and the primary vectors are anthropophilic, sporozoite infection rates are low, typi- cally 10% and often less than 5% [39,40]. Further, infection of mosquitoes even after direct skin feeding on infected falciparum gametocyte-positive volunteers is far from uniform; the average success rate of Anopheles gambiae and Anopheles arabiensis infection from a sur- vey of 930 feeding experiments in a variety of endemic settings was 62% [41]. Accordingly, some proportion of vector mosquitoes consumes blood from infected individuals without becoming infected. To examine the effects of provisioning normal blood histamine (1 nM) and severe malaria-associated blood histamine (10 nM) on uninfected An. stephensi, we first examined the tendency to take a second bloodmeal at 4 d or 14 d later, behavior that Biomolecules 2021, 11, 719 would impact the likelihood of becoming infected at a subsequent bloodmeal. These8 of 23 timepoints reflect the mid-point and completion of the extrinsic incubation period, respectively, for Plasmodium spp. sporogony. Histamine priming had no effect on the tendency tosecond take a bloodmeal second bloodmeal 4 d after 4 thed after first the bloodmeal first bloodmeal (Figure (Figure1). However, 1). However, priming priming with bothwith both1 nM 1 andnM and 10 nM 10 nM histamine histamine was was associated associated with with increased increased tendencies tendencies to to take take aa second bloodmealbloodmeal at at 14 14 d d after after the the first first bloodmeal bloodmeal relative relative to tocontrol control uninfected uninfected An.An. stephensi stephensi ((FigureFigure 22).). TheThe effectseffects ofof bloodblood deliverydelivery ofof histaminehistamine werewere similarsimilar toto thosethose ofof histaminehistamine priming. Specifically, Specifically, blood blood-delivered-delivered histamine histamine had had no no effect effect on on the the tendency tendency to to take take a seconda second bloodmeal bloodmeal 4 d 4 later d later (Figure (Figure 3),3 but), but there there was was an anincreased increased tendency tendency to take to take a sec- a ondsecond bloodmeal bloodmeal at 14 at d 14 later d later in mosquitoes in mosquitoes provisioned provisioned with with 10 10nM nM histamine histamine relative relative to controlto control (Figure (Figure 4).4 To). Tosummarize, summarize, uninfected uninfected mosquitoes mosquitoes responded responded to both to both doses doses of his- of taminehistamine delivered delivered by bypriming priming with with an increased an increased tendency tendency to take to take a second a second bloodmeal bloodmeal at 14 at d14 (Figure d (Figure 2),2 ),while while the the delivery delivery of of histamine histamine in in the the blood blood inc increasedreased this tendency atat 1414 dd atat the treatment dose associated with severe malaria (10 nM; Figure4 4).).

Figure 1. TThehe tendencytendency ofof histamine-primedhistamine-primed uninfecteduninfectedAn. An. stephensi stephensito to take take a a second second bloodmeal bloodmeal 4 d4 later.d later.Left Left: numbers: numbers of offed fed and and unfed unfed uninfected uninfected mosquitoes mosquitoes by treatment.by treatment.Right Rightpanel: panel data: data from from the the left left panel panel shown shown as percentages as percentages of fed of andfed Biomolecules 2021, 11, x FOR PEER REVIEW 9 of 24 andunfed unfed uninfected uninfected mosquitoes mosquitoes in each in each group. group. There There were were no signiﬁcant no significant effects effects of treatment. of treatment. N = N 5 replicates;= 5 replicates; Chi-square Chi-square test (testα = ( 0.05);α = 0.05) hist:; hist: histamine. histamine.

FigureFigure 2. The2. The tendency tendency of of histamine-primed, histamine-primed, uninfected uninfectedAn. An. stephensi stephensito taketo take a second a second bloodmeal bloodmeal 14 14 d later. d later.Left Left: numbers: numbers ofof fed fed and and unfed unfed uninfected uninfected mosquitoes mosquitoes by by treatment treatment with with signiﬁcant significant differences differences noted noted among among pairs. pairs.Right Rightpanel: panel data: data fromfrom the the left left panel panel shown shown as percentages as percentages of fed of andfed unfedand unfed uninfected uninfected mosquitoes mosquitoes in each in group. each group. N = 5; Chi-squareN = 5; Chi-square test (α =test 0.05);(α= hist:0.05) histamine.; hist: histamine. * Control * Control vs. 1 nM vs.p 1= nM 0.034, p = 0.034, control control vs. 10 vs nM. 10p =nM 0.022. p = 0.022.

Figure 3. The tendency of uninfected An. stephensi provisioned with histamine (hist) in blood to take a second bloodmeal 4 d later. Left: numbers of fed and unfed uninfected mosquitoes by treatment. Right panel: data from the left panel shown as percentages of fed and unfed infected mosquitoes in each group. N = 5; Chi-square test (α = 0.05).

Figure 4. The tendency of uninfected An. stephensi provisioned with histamine (hist) in blood to take a second bloodmeal 14 d later. Left: numbers of fed and unfed uninfected mosquitoes by treatment with significant differences noted among pairs. Right panel: data from the left panel shown as percentages of fed and unfed uninfected mosquitoes in each group. N = 5; Chi-square test (α = 0.05). * p = 0.003.

3.2. Histamine Provisioning Enhanced Uninfected Female An. stephensi Flight Activity and Physiological Responses to Olfactory Cues The tendency to take a second bloodmeal is likely to be linked to responses to visual and olfactory cues from the host. To test this directly, we primed female An. stephensi with 10 nM histamine (severe malaria) or diluent (water) in soaked cotton balls for 3 d; sugar cubes were provided as a nutrient source. On the day following priming, we flew the Biomolecules 2021, 11, x FOR PEER REVIEW 9 of 24 Biomolecules 2021, 11, x FOR PEER REVIEW 9 of 24

Figure 2. The tendency of histamine-primed, uninfected An. stephensi to take a second bloodmeal 14 d later. Left: numbers Figure 2. The tendency of histamine-primed, uninfected An. stephensi to take a second bloodmeal 14 d later. Left: numbers of fed and unfed uninfected mosquitoes by treatment with significant differences noted among pairs. Right panel: data Biomoleculesof fed2021 and, 11 unfed, 719 uninfected mosquitoes by treatment with significant differences noted among pairs. Right panel: data9 of 23 from the left panel shown as percentages of fed and unfed uninfected mosquitoes in each group. N = 5; Chi-square test from the left panel shown as percentages of fed and unfed uninfected mosquitoes in each group. N = 5; Chi-square test (α= 0.05); hist: histamine. * Control vs. 1 nM p = 0.034, control vs. 10 nM p = 0.022. (α= 0.05); hist: histamine. * Control vs. 1 nM p = 0.034, control vs. 10 nM p = 0.022.

Figure 3. TThehe tendencytendency of uninfected An.An. stephensistephensi provisionedprovisioned withwith histaminehistamine (hist) (hist) in in blood blood to to take take a a second second bloodmeal bloodmeal 4 Figure 3. The tendency of uninfected An. stephensi provisioned with histamine (hist) in blood to take a second bloodmeal 4d d later. later.Left Left: numbers: numbers of of fed fed and and unfed unfed uninfected uninfected mosquitoes mosquitoes by by treatment. treatment.Right Rightpanel: panel data: data from from the the left left panel panel shown shown as 4 d later. Left: numbers of fed and unfed uninfected mosquitoes by treatment. Right panel: data from the left panel shown as percentages of fed and unfed infected mosquitoes in each group. N = 5; Chi-square testα (α = 0.05). percentagesas percentages of fed of fed and and unfed unfed infected infected mosquitoes mosquitoes in each in each group. grou Np. = N 5; = Chi-square 5; Chi-square test test ( =(α 0.05). = 0.05).

Figure 4. The tendency of uninfected An. stephensi provisioned with histamine (hist) in blood to take a second bloodmeal Figure 4. The tendency of uninfected An. stephensi provisioned with histamine (hist) in blood to take a second bloodmeal 14Figure d later. 4. The Left tendency: numbers of of uninfected fed and unfedAn. stephensi uninfectedprovisioned mosquitoes with by histamine treatment (hist) with in significant blood to take differences a second noted bloodmeal among 14 14 d later. Left: numbers of fed and unfed uninfected mosquitoes by treatment with significant differences noted among pairs.d later. RightLeft: panel numbers: data of from fed and the unfedleft panel uninfected shown mosquitoesas percentages by treatment of fed and with unfed significant uninfected differences mosquitoes noted in amongeach group. pairs. pairs. Right panel: data from the left panel shown as percentages of fed and unfed uninfected mosquitoes in each group. NRight = 5; panel:Chi-square data test from (α the = 0.05). left panel * p = shown0.003. as percentages of fed and unfed uninfected mosquitoes in each group. N = 5; N = 5; Chi-square test (α = 0.05). * p = 0.003. Chi-square test (α = 0.05). * p = 0.003. 3.2. Histamine Provisioning Enhanced Uninfected Female An. stephensi Flight Activity and 3.2. Histamine Provisioning Enhanced Uninfected Female An. stephensi Flight Activity and Physiological Responses to Olfactory Cues 3.2.Physiological Histamine Responses Provisioning to Olfactory Enhanced Cues Uninfected Female An. stephensi Flight Activity and The tendency to take a second bloodmeal is likely to be linked to responses to visual PhysiologicalThe tendency Responses to take to Olfactory a second Cues bloodmeal is likely to be linked to responses to visual and olfactory cues from the host. To test this directly, we primed female An. stephensi with and olfactoryThe tendency cues tofrom take the a secondhost. To bloodmeal test this directl is likelyy, we to primed be linked female to responses An. stephensi to visual with 10 nM histamine (severe malaria) or diluent (water) in soaked cotton balls for 3 d; sugar and10 nM olfactory histamine cues (severe from themalaria) host. or To diluent test this (water) directly, in soaked we primed cotton female balls An.for 3 stephensid; sugar cubes were provided as a nutrient source. On the day following priming, we flew the withcubes 10 were nM provided histamine as (severe a nutrient malaria) source. or diluentOn the (water)day following in soaked priming, cotton we balls flew for the 3 d; sugar cubes were provided as a nutrient source. On the day following priming, we flew the mosquitoes in a wind tunnel (Figure5A) that allowed control of the airflow conditions (olfactory stimuli) and visual stimuli that mosquitoes might experience in nature. The occupancy map showed that during clean air treatment, few mosquitoes flew in the tunnel (Figure5B , n = 372 for this replicate, top panel; n = 1944 total for all replicates). By contrast, CO2 exposure was associated with greater numbers of mosquitoes flying in the tunnel (Figure5B , n = 1529 for this replicate, bottom panel; n = 6182 total for all replicates). In control and primed groups, the proportion investigating visual objects increased significantly from 1%–2% in clean air to 5%–6% during CO2 exposure (gray vs black bars, Figure5C), but priming did not increase this behavior in CO 2-exposed mosquitoes (control black bar vs histamine-primed black bar, Figure5C). Priming, however, significantly increased CO2-induced flight activity compared to controls (control black bar vs histamine-primed black bar, Figure5D). CO 2 significantly increased the flight velocities of both control and 10 nM histamine-primed mosquitoes relative to clean air (gray vs black bars, Figure5E), but priming did not increase this behavior in CO 2-exposed mosquitoes Biomolecules 2021, 11, 719 10 of 23

Biomolecules 2021, 11, x FOR PEER REVIEW 11 of 24 (control black bar vs histamine-primed black bar, Figure5E). In all studies, the behavior returned to the baseline post-CO2, p > 0.05.

Figure 5. The flightFigure activity 5. The of flightAn. stephensiactivity ofand An. the stephensi response and to visualthe response objects to with visua histaminel objects (hist) with priminghistamine or (hist) provisioning in a bloodmeal.priming (A) Wind or tunnelprovisioning with real-time in a bloodmeal. tracking to(A quantify) Wind tunnel mosquito with behavior real-time to tracking olfactory to and quantify visual mos- cues. (Right) Top and side viewsquito of behavior a mosquito to olfactory trajectory and investigating visual cues. the (Right) visual Top object and (black side views circle). of ( Ba) mosquito Occupancy trajectory maps of mosquito investigating the visual object (black circle). (B) Occupancy maps of mosquito activity during fil- activity during filtered air (top) and CO2 exposure (bottom). (C–E) Investigation of visual objects (C), flight activity (D), tered air (top) and CO2 exposure (bottom). (C–E) Investigation of visual objects (C), flight activity and flight velocities (E) of histamine-primed and control mosquitoes to filtered air and CO2 treatments. * p < 0.05 (Wilcoxon (D), and flight velocities (E) of histamine-primed and control mosquitoes to filtered air and CO2 rank test for C and D, t-test for E); (ns) denotes not significantly different (p > 0.05). (F–H) Mosquitoes provisioned with treatments. * p < 0.05 (Wilcoxon rank test for C and D, t-test for E); (ns) denotes not significantly 10 nM histaminedifferent in a bloodmeal (p > 0.05). or (F control–H) Mosquitoes mosquitoes provisioned (no histamine with in10 thenM bloodmeal) histamine in and a bloodmeal their investigation or control of visual objects (F), flightmosquitoes activity (G (no), and histamine flight velocities in the bloodme (H). * pal)< 0.05and (Wilcoxontheir investigation rank test of for visualF and objectsG; t-test (F), for flightH); (ns)ac- denotes not significantlytivity different (G), and (p > flight 0.05). velocities (H). * p < 0.05 (Wilcoxon rank test for F and G; t-test for H); (ns) denotes not significantly different (p > 0.05). We conducted similar experiments using An. stephensi provisioned with 10 nM his- To determinetamine if in behavioral an RBC-serum responses meal were or an ass RBC-serumociated with meal changes supplemented in sensory with phys- an equivalent iology, we electrophysiologicallyvolume of water usedas recorded histamine from diluent. the antenna For both and control eye andof ahistamine-fed randomly se- mosquitoes, lected sampleCO of2 increasedmosquitoes the from number behavioral of mosquitoes experiments investigating in Figure 5. the We visual completed objects elec- (gray vs black troantennogrambars, Figure(EAG) 5andF),but low histamine light (70 treatmentμW/cm2/nm) did electroretinogram not increase this behavior(ERG) record- in CO 2-exposed ings, outputsmosquitoes that are thought (control to black reflect bar the vs sum histamine-fed of receptor blackneuron bar, responses Figure5F). of Thethe an- CO 2-induced tenna and eye,flight respectively. activity relative Histamine to clean-primed air was mosquitoes significantly exhibited increased significantly in both control greater and histamine- EAG responsestreated to odor mosquitoes stimulation (gray than vs black control bars, mo Figuresquitoes5G), (Figure but histamine 6A). While treatment histamine did not- increase primed, histaminethis behavior blood- infed CO, and2-exposed control mosquitoesmosquitoes (control exhibited black significant bar vs histamine-fed ERG responses black bar, Fig- to the testedure wavelengths5G). Like histamine-primed (Kruskal–Wallis mosquitoes, test, p < 0.01), the flightwith velocitythe greatest of CO response2-exposed to mosquitoes green (530 nm),was notfollowed increased by white, by histamine blue (460 provisioningnm), and red in(635 an nm) RBC-serum relative to meal a black relative no- to control stimulus controlmosquitoes (Figure (control 6B), histamine black bar delivery vs histamine-fed via priming black or RBC bar,-serum Figure 5mealH). Overall,did not histamine increase these responses relative to control. To place these sensory physiological responses in a tissue context consistent with ingestion of histamine, we confirmed histamine staining (Figure 6C) in the midgut, thoracic ganglion, and visual and olfactory regions of Biomolecules 2021, 11, 719 11 of 23

priming and provisioning did not increase the investigation of visual objects (Figure5C,F) or flight velocities of CO2-exposed mosquitoes (Figure5E,H), but flight activities of CO 2- exposed mosquitoes relative to controls were significantly increased in primed mosquitoes (Figure5D) and trended toward an increase in provisioned mosquitoes (Figure5G). To determine if behavioral responses were associated with changes in sensory physiology, we electrophysiologically recorded from the antenna and eye of a randomly selected sample of mosquitoes from behavioral experiments in Figure5. We completed electroantennogram (EAG) and low light (70 µW/cm2/nm) electroretinogram (ERG) recordings, outputs that are thought to reflect the sum of receptor neuron responses of the antenna and eye, respectively. Histamine-primed mosquitoes exhibited significantly greater EAG responses to odor stimulation than control mosquitoes (Figure6A). While histamine-primed, histamine blood-fed, and control mosquitoes exhibited significant ERG responses to the tested wavelengths (Kruskal–Wallis test, p < 0.01), with the greatest response to green (530 nm), followed by white, blue (460 nm), and red (635 nm) relative to a black no-stimulus control (Figure6B), histamine delivery via priming or RBC-serum meal did not increase these responses relative to control. To place these sensory physiological responses in a tissue context consistent with ingestion of histamine, we confirmed histamine staining (Fig- ure6C) in the midgut, thoracic ganglion, and visual and olfactory regions of the brain. The midgut showed strong staining in the interior (Figure6C, arrow, left panel). The thoracic ganglion showed bleb-like tracts throughout the ganglion (Figure6C, arrow, middle panel), while the brain showed strong staining in loci associated with the antennal lobe (AL) and optic lobe (OL) (Figure6C, arrows, right panel). Taken together, our behavioral and electrophysiological assays suggested an association between increased olfactory responsiveness (Figure6A) and increased flight activity in CO 2-exposed, histamine-primed mosquitoes (Figure5D). Histamine treatment, however, had no effect in CO 2-exposed mosquitoes on the response to visual cues relative to controls (Figure5C,F), and this correlated with a lack of histamine-enhanced ERG responses (Figure6B).

3.3. Histamine Provisioning Had Modest to No Effects on Uninfected Female An. stephensi Lifespan, But Increased the Tendency to Take Weekly Bloodmeals with Increasing Age A recent study of An. stephensi suggested that mosquito survival to 35–60 days occurs across a broad temperature range [42]. The authors also showed that the proportion of females blood-feeding declined with increasing mosquito age, so an increase in feeding tendency at a more advanced age promoted by ingested histamine could partially offset age-related declines in blood-feeding to promote parasite infection and transmission. Based on these observations and the fact that mosquito longevity is perhaps the most important variable for vectorial capacity, we examined the survival of female An. stephensi that were offered weekly RBC-serum meals supplemented with 1 nM or 10 nM histamine or RBC- serum meals supplemented with a volume of water equivalent to that used for the addition of histamine. In one replicate, mosquitoes that received weekly meals supplemented with 10 nM histamine lived signiﬁcantly longer than controls (Figure7A), with increased survival apparent beyond 42 d. In a second replicate (Figure7B), no differences in survival were detected, afﬁrming that histamine effects on An. stephensi lifespan are modest to negligible. Given that blood-feeding tendency with increasing age directly impacts parasite transmission, we also examined the proportion of mosquitoes in each group that blood-fed each week (Figure8). The proportional feeding data from these replicates were combined for analysis because the Kaplan–Meier test showed no difference (p = 0.0631) between mean times to failure to feed for the control in replicate 1 (23.04 days) and the control in replicate 2 (26.62 days). From weeks 2–6, a greater proportion of mosquitoes that were provisioned with 10 nM histamine fed on the RBC-serum meal compared with mosquitoes that were provisioned with 1 nM histamine each week (Figure8). These results indicate that weekly exposure to 10 nM histamine can increase the tendency of An. stephensi to bloodfeed even at extended to ages in a manner that is not dependent on changes in survivorship. Biomolecules 2021, 11, x FOR PEER REVIEW 12 of 24

the brain. The midgut showed strong staining in the interior (Figure 6C, arrow, left panel). The thoracic ganglion showed bleb-like tracts throughout the ganglion (Figure 6C, arrow, middle panel), while the brain showed strong staining in loci associated with the antennal lobe (AL) and optic lobe (OL) (Figure 6C, arrows, right panel). Taken together, our behavioral and electrophysiological assays suggested an association between increased olfactory responsiveness (Figure 6A) and increased flight activity in CO2-exposed, histamine- primed mosquitoes (Figure 5D). Histamine treatment, however, had no effect in CO2-ex- Biomolecules 2021, 11, 719 12 of 23 posed mosquitoes on the response to visual cues relative to controls (Figure 5C,F), and this correlated with a lack of histamine-enhanced ERG responses (Figure 6B).

FigureFigure 6. 6. ElectrophysiologicalElectrophysiological recordings recordings and and histaminergic histaminergic innervation innervation of An. of An. steph stephensiensi tissues.tissues. (A(A) )Histamine Histamine (hist) (hist) priming priming caused caused increased increased EAG responsesresponses ((nn == 15)15) thatthat werewere signiﬁcantlysignificantly greater greater than control (t-test: * p = 0.04). The traces show the mean response for the two treatment than control (t-test: * p = 0.04). The traces show the mean response for the two treatment groups (left); groups (left); the bars are means ± SE (right). (B) ERG responses (n = 30) to different light stimuli. the bars are means ± SE (right). (B) ERG responses (n = 30) to different light stimuli. Responses were Responses were not significantly different between histamine-treated (primed and blood-fed) and controlnot signiﬁcantly groups (t-test: different p > 0.05). between Traces histamine-treated are responses to the (primed light stimuli and blood-fed) for the primed and control mosquitoes groups (t-test: p > 0.05). Traces are responses to the light stimuli for the primed mosquitoes (left, top) and blood-fed mosquitoes (left, bottom). Bars are the means ± SE (right). (C) Schematic of An. stephensi tissues (midgut, thoracic ganglion, brain) that were immunolabeled for histamine. The white arrows denote the strong histamine staining (red) of the midgut lining (left, bottom) and neuropil in the thoracic ganglion (middle, bottom). Section of the brain shows strong immunolabeling of histamine in cell clusters immediately anterior to the antennal lobe (AL) and cells adjacent to the olfactory lobe (OL). White lines denote 100 µm distance. Biomolecules 2021, 11, x FOR PEER REVIEW 13 of 24

(left, top) and blood-fed mosquitoes (left, bottom). Bars are the means ± SE (right). (C) Schematic of An. stephensi tissues (midgut, thoracic ganglion, brain) that were immunolabeled for histamine. The white arrows denote the strong histamine staining (red) of the midgut lining (left, bottom) and neuropil in the thoracic ganglion (middle, bottom). Section of the brain shows strong immunolabeling of histamine in cell clusters immediately anterior to the antennal lobe (AL) and cells adjacent to the olfactory lobe (OL). White lines denote 100 μm distance.

3.3. Histamine Provisioning Had Modest to No Effects on Uninfected Female An. stephensi Lifespan, But Increased the Tendency to Take Weekly Bloodmeals with Increasing Age A recent study of An. stephensi suggested that mosquito survival to 35–60 days occurs across a broad temperature range [42]. The authors also showed that the proportion of females blood-feeding declined with increasing mosquito age, so an increase in feeding tendency at a more advanced age promoted by ingested histamine could partially offset age-related declines in blood-feeding to promote parasite infection and transmission. Based on these observations and the fact that mosquito longevity is perhaps the most important variable for vectorial capacity, we examined the survival of female An. stephensi that were offered weekly RBC-serum meals supplemented with 1 nM or 10 nM histamine or RBC-serum meals supplemented with a volume of water equivalent to that used for the addition of histamine. In one replicate, mosquitoes that received weekly meals supplemented with 10 nM histamine lived significantly longer than controls (Figure 7A), with increased survival apparent beyond 42 d. In a second replicate (Figure 7B), no differences in survival were detected, affirming that histamine effects on An. stephensi lifespan are modest to negligible. Given that blood-feeding tendency with increasing age directly impacts parasite transmission, we also examined the proportion of mosquitoes in each group that blood-fed each week (Figure 8). The proportional feeding data from these replicates were combined for analysis because the Kaplan–Meier test showed no difference (p = 0.0631) between mean times to failure to feed for the control in replicate 1 (23.04 days) and the control in replicate 2 (26.62 days). From weeks 2–6, a greater proportion of mosquitoes that were provisioned with 10 nM histamine fed on the RBC-serum meal compared with mosquitoes that were provisioned with 1 nM histamine each week (Figure 8). These re- Biomolecules 2021, 11, 719 sults indicate that weekly exposure to 10 nM histamine can increase the tendency of13 An. of 23 stephensi to bloodfeed even at extended to ages in a manner that is not dependent on changes in survivorship.

FigureFigure 7. 7. PercentPercent survival survival of ofuninfected uninfected mosquitoes mosquitoes provisioned provisioned with with 1 nM 1 histamine nM histamine (hist) (hist) and 10 and nM 10 histamine nM histamine or with or anwith equivalent an equivalent volume volume of water of waterdiluent diluent (control) (control) in a weekly in a weekly bloodmeal. bloodmeal. (A) Replicate (A) Replicate 1. The 1. survival The survival of mosquitoes of mosquitoes provisionedprovisioned with with10 nM 10 histamine nM histamine was significantly was signiﬁcantly different different from the from survival the survival of control of control mosquitoes mosquitoes and mosquitoes and mosquitoes provisioned with 1 nM histamine. Kaplan–Meier with log-rank test (α = 0.05); control vs 10 nM histamine, p = 0.0242. (B) Rep- Biomoleculesprovisioned 2021, 11, with x FOR 1 PEER nM histamine. REVIEW Kaplan–Meier with log-rank test (α = 0.05); control vs 10 nM histamine, p = 0.0242.14 (B )of 24 licate 2. There were no significant differences in survival among treatments. Kaplan–Meier with log-rank test (α = 0.05). Replicate 2. There were no signiﬁcant differences in survival among treatments. Kaplan–Meier with log-rank test (α = 0.05).

FigureFigure 8. 8. PercentPercent feeding feeding of ofuninfected uninfected mosquitoes mosquitoes provisioned provisioned with with 1 nM 1 nMhistamine histamine or 10 or nM 10 nM histaminehistamine in in weekly bloodmeals overover time.time. AA greatergreater proportion proportion of of mosquitoes mosquitoes that that were were provisioned provisioned with 10 nM histamine fed on the RBC-serum meal compared with mosquitoes that were with 10 nM histamine fed on the RBC-serum meal compared with mosquitoes that were provisioned provisioned with 1 nM histamine each week. N = 2; Wilcoxon test (α = 0.05), 1 nM vs 10 nM hista- with 1 nM histamine each week. N = 2; Wilcoxon test (α = 0.05), 1 nM vs 10 nM histamine, p = 0.0189. mine, p = 0.0189. 3.4. Histamine Provisioning Enhanced An. stephensi Infection with P. y. yoelii 17XNL 3.4. Histamine Provisioning Enhanced An. stephensi Infection with P. y. yoelii 17XNL The tendency for a mosquito to become infected is obviously critical for transmission, but givenThe tendency the fact thatfor a some mosquito studies to become have suggested infected thatis obviously sporozoite critical density for andtransmission, infectivity butdecline given over the thefact firstthat foursome weeks studies of have salivary suggested gland infectionthat sporozoite [43], the density intensity and of infectivity infection declineshould over not bethe overlooked first four weeks in considering of salivary the gland likelihood infection of [43] transmission, the intensity with of increasing infection shouldmosquito not age.be overlooked For these in studies, considering we used the CD1likelihood outbred of transmission mice, which with have increasing low basal mosquitoplasma histamine age. For these levels studies, (20–30 nM we [used44]) andCD1 do outbred not exhibit mice, increased which have circulating low basal histamine plasma histaminein the peripheral levels (20 blood–30 nM in [44] response) and do to notP. y.exhibit yoelii increased17XNL infectioncirculating (Figure histamine S1). in With the peripheralhistamine priming,blood in theresponse proportions to P. ofy. mosquitoesyoelii 17XNL with infection at least (Figure one oocyst S1). perWith midgut histamine were priming,significantly the proportions higher in the of 10mosquitoes nM histamine with at group least comparedone oocyst to per both midgut control were and signifi- 1 nM cantlyhistamine-treated higher in the mosquitoes 10 nM histamine (Figure group9). Further, compared histamine to both priming control and was 1 associated nM histamine with- treatedsignificantly mosquitoes increased (Figure mean 9). oocysts Further, per hiinfectedstamine midgut priming compared was associated to controls with (Figure signifi- 10). cantlyHistamine increased priming mean had oocysts no effect per on infected the proportions midgut compared of mosquitoes to controls with ( infectedFigure 10). salivary His- tamineglands priming(Figure 11 had), but no salivary effect on gland the sporozoite proportions density of mosquitoes was significantly with infected increased salivary with glands10 nM ( histamineFigure 11), priming but salivary relative gland to controls sporozoite (Figure density 12). was significantly increased with 10nM histamine priming relative to controls (Figure 12).

Figure 9. Proportions of An. stephensi infected with P. y. yoelii 17XNL oocysts following priming for 3 d prior to infection with histamine (hist) in water or water only via soaked cotton balls. Left: numbers of infected and uninfected mosquitoes by treatment with significant differences noted among pairs. Right panel: data from the left panel shown as percentages of uninfected and infected mosquitoes in each group. N = 10; Chi-square test (α = 0.05). *** p = 4.6 × 10−5, ** p = 0.0016. Biomolecules 2021, 11, x FOR PEER REVIEW 14 of 24

Figure 8. Percent feeding of uninfected mosquitoes provisioned with 1 nM histamine or 10 nM histamine in weekly bloodmeals over time. A greater proportion of mosquitoes that were provisioned with 10 nM histamine fed on the RBC-serum meal compared with mosquitoes that were provisioned with 1 nM histamine each week. N = 2; Wilcoxon test (α = 0.05), 1 nM vs 10 nM histamine, p = 0.0189.

3.4. Histamine Provisioning Enhanced An. stephensi Infection with P. y. yoelii 17XNL The tendency for a mosquito to become infected is obviously critical for transmission, but given the fact that some studies have suggested that sporozoite density and infectivity decline over the first four weeks of salivary gland infection [43], the intensity of infection should not be overlooked in considering the likelihood of transmission with increasing mosquito age. For these studies, we used CD1 outbred mice, which have low basal plasma histamine levels (20–30 nM [44]) and do not exhibit increased circulating histamine in the peripheral blood in response to P. y. yoelii 17XNL infection (Figure S1). With histamine priming, the proportions of mosquitoes with at least one oocyst per midgut were significantly higher in the 10 nM histamine group compared to both control and 1 nM histamine- treated mosquitoes (Figure 9). Further, histamine priming was associated with significantly increased mean oocysts per infected midgut compared to controls (Figure 10). His- Biomolecules 2021, 11, 719 tamine priming had no effect on the proportions of mosquitoes with infected salivary14 of 23 glands (Figure 11), but salivary gland sporozoite density was significantly increased with 10nM histamine priming relative to controls (Figure 12).

FigureFigure 9. 9.Proportions Proportions of ofAn. An. stephensi stephensiinfected infected with withP. P. y. y. yoelii yoelii17XNL 17XNL oocysts oocysts following following priming priming for for 3 3 d d prior prior to to infection infection withwith histamine histamine (hist) (hist) in in water water or or water water only only via via soaked soaked cotton cotton balls. balls.Left Left:: numbers numbers of of infected infected and and uninfected uninfected mosquitoes mosquitoes Biomoleculesbyby treatment treatment 2021, 11, with xwith FOR signiﬁcant significantPEER REVIEW differences differences noted noted among among pairs. pairs.Right Rightpanel: panel data: data from from the the left left panel panel shown shown as percentagesas percentages15 of of 24 −5 Biomoleculesuninfectedof uninfected 2021, 11 and, x andFOR infected infectedPEER mosquitoes REVIEW mosquitoe ins each in each group. group. N = N 10; = Chi-square10; Chi-square test test (α = (α 0.05). = 0.05). *** p***= p 4.6 = 4.6× 10 × −105, **, **p p= = 0.0016. 0.0016. 15 of 24

Figure 10. TheThe m meanean P.P. y. y. yoelii yoelii 17XNL17XNL midgut midgut oocysts oocysts in in An.An. stephensi stephensi followingfollowing priming priming for for 3 d 3 d Figureprior to 10. infection The mean with P. histamine y. yoelii 17XNL (hist) (hist) midgutor or water water oocysts (control) (control) in An.via via soakedstephensi soaked cotton cotton following pads. pads. priming N N == 10; 10; onefor one-way -3way d priorANOVA to infection (α = 0.05). with **** histamine p-value = (hist) 0.00012, or water * p = 0.042.(control) via soaked cotton pads. N = 10; one-way ANOVA (α = 0.05). **** p-value = 0.00012, * p = 0.042. ANOVA (α = 0.05). **** p-value = 0.00012, * p = 0.042.

Figure 11. Proportions of An. stephensi with P. y. yoelii 17XNL salivary gland sporozoites following priming for 3 d prior FigureFigureto infection 11. Proportions with histamine of of An. (hist) stephensi stephensi or wa terwithwith (control) P.P. y. yoelii via 17XNLsoaked cottonsalivary pads. gland Left sporozoites : numbers followingof following mosquitoes priming priming with for forinfected 3 3 d d prior and totouninfected infection infection withsalivary with histamine histamine glands. (hist) (hist)Right or or panelwa waterter: (control)data (control) from via the via soaked left soaked panel cotton cotton shown pads. pads. as Left percentagesLeft: numbers: numbers of mosquitoesuninfected of mosquitoes andwith withinfected infected infected mos- and quitoes in each group. N = 6; Chi-square test (α = 0.05), no significance. uninfectedand uninfected salivary salivary glands. glands. RightRight panelpanel:: data from data fromthe left the panel left panelshown shown as percentages as percentages of uninfected of uninfected and infected and infected mosquitoes in each group. N = 6; Chi-square test (α = 0.05), no significance. mosquitoes in each group. N = 6; Chi-square test (α = 0.05), no signiﬁcance.

Figure 12. The mean salivary gland score in An. stephensi infected with P. y. yoelii 17XNL sporozo- Figureites following 12. The primingmean salivary for 3 d gland prior scoreto infection in An. withstephensi histamine infected (hist) with or P. water y. yoelii (control) 17XNL via sporozo- soaked itescotton following pads. N priming = 6; one for-way 3 d ANOVA prior to infection(α = 0.05). with * p-value histamine = 0.00484. (hist) or water (control) via soaked cotton pads. N = 6; one-way ANOVA (α = 0.05). * p-value = 0.00484. Biomolecules 2021, 11, x FOR PEER REVIEW 15 of 24

Figure 10. The mean P. y. yoelii 17XNL midgut oocysts in An. stephensi following priming for 3 d prior to infection with histamine (hist) or water (control) via soaked cotton pads. N = 10; one-way ANOVA (α = 0.05). **** p-value = 0.00012, * p = 0.042.

Figure 11. Proportions of An. stephensi with P. y. yoelii 17XNL salivary gland sporozoites following priming for 3 d prior Biomoleculesto infection2021, 11, with 719 histamine (hist) or water (control) via soaked cotton pads. Left: numbers of mosquitoes with infected and15 of 23 uninfected salivary glands. Right panel: data from the left panel shown as percentages of uninfected and infected mosquitoes in each group. N = 6; Chi-square test (α = 0.05), no significance.

Biomolecules 2021, 11, x FOR PEER REVIEW 16 of 24 FigureFigure 12. 12.The The mean mean salivary salivary gland gland score score in inAn. An. stephensi stephensiinfected infected with withP. y.P. yoeliiy. yoelii17XNL 17XNL sporozoites sporozoites following priming for 3 d prior to infection with histamine (hist) or water (control) via soaked following priming for 3 d prior to infection with histamine (hist) or water (control) via soaked cotton cotton pads. N = 6; one-way ANOVA (α = 0.05). * p-value = 0.00484. pads. N = 6; one-way ANOVA (α = 0.05). * p-value = 0.00484. 3.5. Histamine Provisioning Had No Effect on the Tendency of P. y. yoelii 17XNL-Infected An. stephensi3.5. Histamine to Take Provisioning a Subsequent Had Bloodmeal No Effect on the Tendency of P. y. yoelii 17XNL-Infected An. stephensi to Take a Subsequent Bloodmeal The tendency to take a second bloodmeal by an infected mosquito is of obvious im- portanceThe for tendency transmission. to take However, a second the bloodmeal timing of by the an second infected meal mosquito is critical. is ofIf the obvious meal importance for transmission. However, the timing of the second meal is critical. If the is taken during oocyst infection, there is a risk that the mosquito could die in the bout with meal is taken during oocyst infection, there is a risk that the mosquito could die in the bout no resulting parasite transmission. Accordingly, we selected two time points for these with no resulting parasite transmission. Accordingly, we selected two time points for these studies: 4 d PI when only oocysts were present or 11 d PI when sporozoites were present studies: 4 d PI when only oocysts were present or 11 d PI when sporozoites were present in in the glands. Histamine priming had no effect on the tendency to take a second blood- the glands. Histamine priming had no effect on the tendency to take a second bloodmeal meal by infected An. stephensi at either 4 d or 11 d PI (Figures 13 and 14). Combined with by infected An. stephensi at either 4 d or 11 d PI (Figures 13 and 14). Combined with the data above, ingestion of elevated levels of histamine (10 nM) associated with severe the data above, ingestion of elevated levels of histamine (10 nM) associated with severe malaria increased the tendency of uninfected An. stephensi to take a second bloodmeal and malaria increased the tendency of uninfected An. stephensi to take a second bloodmeal and increased midgut and salivary gland infection success with P. y. yoelii 17XNL, but in the increased midgut and salivary gland infection success with P. y. yoelii 17XNL, but in the presence of parasite infection, histamine provisioning did not increase the tendency to presence of parasite infection, histamine provisioning did not increase the tendency to take take a second bloodmeal. a second bloodmeal.

Figure 13.13. Tendency ofof histamine-primedhistamine-primed An. stephensi infected with P. y.y. yoeliiyoelii 17XNL17XNL to taketake aa secondsecond bloodmealbloodmeal at 4 dd post-infectionpost-infection (PI).(PI). LeftLeft:: numbers ofof fed and unfed infected mosquitoesmosquitoes byby treatment.treatment. RightRight panel:panel: data from the left panel shownshown asas percentagespercentages ofof fedfed andand unfedunfed infectedinfected mosquitoesmosquitoes in in each each group. group. NN == 5;5; Chi-squareChi-square testtest ((αα == 0.05);0.05); hist: histamine.

Figure 14. Tendency of histamine-primed A. stephensi infected with P. y. yoelii 17XNL to take a second bloodmeal at 11 d post-infection (PI). Left: numbers of fed and unfed infected mosquitoes by treatment. Right panel: data from the left panel shown as percentages of fed and unfed infected mosquitoes in each group. N = 5; Chi-square test (α = 0.05); hist: histamine.

3.6. Histamine Provisioning Enhanced An. stephensi Infection with P. falciparum NF54 Strain To establish whether observations with a mouse malaria parasite could be extended to infection with a human parasite, we examined the infection success of An. stephensi using cultured P. falciparum NF54. In vitro culture of this parasite also allowed us to pose an important question: could malaria parasites respond directly to histamine, suggesting that histamine might concurrently alter both mosquito and parasite biology during infection of the mosquito host? To address this question, we treated parasites in a culture based on our established drug assay protocol [32,45–48], which we modified for the use of Biomolecules 2021, 11, x FOR PEER REVIEW 16 of 24

3.5. Histamine Provisioning Had No Effect on the Tendency of P. y. yoelii 17XNL-Infected An. stephensi to Take a Subsequent Bloodmeal The tendency to take a second bloodmeal by an infected mosquito is of obvious importance for transmission. However, the timing of the second meal is critical. If the meal is taken during oocyst infection, there is a risk that the mosquito could die in the bout with no resulting parasite transmission. Accordingly, we selected two time points for these studies: 4 d PI when only oocysts were present or 11 d PI when sporozoites were present in the glands. Histamine priming had no effect on the tendency to take a second bloodmeal by infected An. stephensi at either 4 d or 11 d PI (Figures 13 and 14). Combined with the data above, ingestion of elevated levels of histamine (10 nM) associated with severe malaria increased the tendency of uninfected An. stephensi to take a second bloodmeal and increased midgut and salivary gland infection success with P. y. yoelii 17XNL, but in the presence of parasite infection, histamine provisioning did not increase the tendency to take a second bloodmeal.

BiomoleculesFigure2021 13., 11 Tendency, 719 of histamine-primed An. stephensi infected with P. y. yoelii 17XNL to take a second bloodmeal at 416 d of 23 post-infection (PI). Left: numbers of fed and unfed infected mosquitoes by treatment. Right panel: data from the left panel shown as percentages of fed and unfed infected mosquitoes in each group. N = 5; Chi-square test (α = 0.05); hist: histamine.

FigureFigure 14. 14.Tendency Tendency of of histamine-primed histamine-primedA. A. stephensi stephensiinfected infected with withP. P. y. y. yoelii yoelii17XNL 17XNL to to take take a a second second bloodmeal bloodmeal at at 11 11 d d post-infectionpost-infection (PI). (PI).Left Left: numbers: numbers of of fed fed and and unfed unfed infected infected mosquitoes mosquitoes by by treatment. treatment.Right Rightpanel: panel data: data from from the the left left panel panel shown as percentages of fed and unfed infected mosquitoes in each group. N = 5; Chi-square test (α = 0.05); hist: histamine. shown as percentages of fed and unfed infected mosquitoes in each group. N = 5; Chi-square test (α = 0.05); hist: histamine. 3.6. Histamine Provisioning Enhanced An. stephensi Infection with P. falciparum NF54 Strain 3.6. HistamineTo establish Provisioning whether Enhancedobservations An. stephensiwith a mouse Infection malaria with P.parasite falciparum could NF54 be extended Strain to infectionTo establish with whether a human observations parasite, we with examined a mouse malariathe infection parasite success could of be An. extended stephensi to infectionusing cultured with a humanP. falciparum parasite, NF54. we In examined vitro culture the infection of this parasite success also of An. allowed stephensi us tousing pose culturedan importantP. falciparum question:NF54. couldIn malaria vitro culture parasites of thisrespond parasite directly also to allowed histamine, us to suggesting pose an importantthat histamine question: might could concurrently malaria parasites alter both respond mosquito directly and parasite to histamine, biology suggesting during infec- that histaminetion of the might mosquito concurrently host? To address alter both this mosquito question, and we parasitetreated parasites biology duringin a culture infection based ofon the our mosquito established host? drug To address assay protocol this question, [32,45– we48], treated which parasites we modified in a culture for the based use of on our established drug assay protocol [32,45–48], which we modified for the use of Hoechst and JC-1 stains versus propidium iodide. Based on the results of these assays, we confirmed that P. falciparum growth is not directly altered by histamine at the concentrations used in our assays (Figure 15). While asexual parasites in culture are not biologically equivalent to mosquito-stage parasites, the lack of encoded receptors for histamine in the genomes of Plasmodium spp. supports our assumption that histamine treatment of infected An. stephensi alters mosquito biology only. For parasite infection studies, female An. stephensi were allowed to feed on gametocyte culture, to which histamine was added to final concentrations of 1 nM or 10 nM immediately before blood-feeding. An aliquot of the same parasite culture was supplemented as a control with a volume of water equivalent to that used to add the histamine treatments. With histamine provisioning, the proportion of mosquitoes with at least one oocyst per midgut was significantly increased in the 10 nM histamine group compared to control mosquitoes (Figure 16). Priming with 10 nM histamine was also associated with increased mean oocysts per infected midgut compared to control mosquitoes (Figure 17). The proportion of mosquitoes with salivary gland sporozoites was significantly higher with provisioning of 10 nM histamine compared to both control and 1 nM histamine treated mosquitoes (Figure 18), but there was no effect on sporozoite density, as all were a score of 1. Together with the observations above, these data suggest that elevated histamine (10 nM) associated with severe malaria promotes infection of a single mosquito host with both P. y. yoelii 17XNL and P. falciparum parasites with notably divergent genetics and life cycle biology [49]. Biomolecules 2021, 11, x FOR PEER REVIEW 17 of 24

Hoechst and JC-1 stains versus propidium iodide. Based on the results of these assays, we confirmed that P. falciparum growth is not directly altered by histamine at the concentrations used in our assays (Figure 15). While asexual parasites in culture are not biologically equivalent to mosquito-stage parasites, the lack of encoded receptors for histamine in the genomes of Plasmodium spp. supports our assumption that histamine treatment of infected An. stephensi alters mosquito biology only. For parasite infection studies, female An. stephensi were allowed to feed on gametocyte culture, to which histamine was added to final concentrations of 1 nM or 10 nM immediately before blood-feeding. An aliquot of the same parasite culture was supplemented as a control with a volume of water equivalent to that used to add the histamine treatments. With histamine provisioning, the proportion of mosquitoes with at least one oocyst per midgut was significantly increased in the 10 nM histamine group compared to control mosquitoes (Figure 16). Priming with 10 nM histamine was also associated with increased mean oocysts per infected midgut compared to control mosquitoes (Figure 17). The proportion of mosquitoes with salivary gland sporozoites was significantly higher with provisioning of 10 nM histamine compared to both control and 1 nM histamine treated mosquitoes (Figure 18), but there was no effect on sporozoite density, as all were a score of 1. Together with the observations above, these data suggest that elevated histamine (10 nM) associated with severe malaria promotes Biomolecules 2021, 11, 719 infection of a single mosquito host with both P. y. yoelii 17XNL and P. falciparum parasites17 of 23 with notably divergent genetics and life cycle biology [49].

FigureFigure 15. 15. HistamineHistamine treatment treatment of P. of falciparumP. falciparum NF54NF54 in vitroin vitro had nohad significant no significant direct directeffects effects on on parasiteparasite growth. growth. LeftLeft: :percent percent of of parasitemias parasitemias at at48 48 h h(one (one growth growth cycle) cycle) following following treatment treatment with with 1, 1,10, 10 and, and 100 100 nM nM histamine histamine (hist), (hist), an an equivalent equivalent volume volume of of water water used used toto deliverdeliver histaminehistamine treatmentstreat- mentsor only or mediaonly media added added to parasites. to parasites. Human Human RBCs RBCs were were included included to to indicate indicate the the specificity specificity ofof flow flow cytometric detection of fluorescence. Right panel: parasites from the left panel at 96 h after Biomolecules 2021, 11, x FOR PEER REVIEWcytometric detection of fluorescence. Right panel: parasites from the left panel at 96 h after treatment.18 of 24 Biomolecules 2021, 11, x FOR PEER REVIEWtreatment. N = 4 replicates analyzed per treatment, and each analyzed in duplicate; ANOVA 18(α =of 24 α 0.05).N =4 replicates analyzed per treatment, and each analyzed in duplicate; ANOVA ( = 0.05).

FigureFigure 16. 16.Proportions Proportions of ofAn. An. stephensi stephensiinfected infected withwith P.P. falciparumfalciparum NF54NF54 oocysts with histamine (hist) (hist) provisioning provisioning in in the the Figure 16. Proportions of An. stephensi infected with P. falciparum NF54 oocysts with histamine (hist) provisioning in the infected bloodmeal. Left: numbers of infected and uninfected mosquitoes by treatment with significant differences noted infectedinfected bloodmeal. bloodmeal.Left Left: numbers: numbers of of infected infected andand uninfecteduninfected mosquitoes by treatment treatment with with significant signiﬁcant differences differences noted noted among pairs. Right panel: data from the left panel shown as percentages of uninfected and infected mosquitoes in each amongamong pairs. pairs.Right Rightpanel: panel data: data from from the the left left panel panel shownshown asas percentagespercentages of uninfected and and infected infected mosquitoes mosquitoes in in each each group. N = 4; Chi-square test (α = 0.05). * p = 0.0108. group.group. N N = = 4; 4; Chi-square Chi-square test test ( α(α= =0.05). 0.05). ** pp == 0.0108.

Figure 17. Mean P. falciparum NF54 midgut oocysts in An. stephensi with histamine (hist) provi- Figure 17. Mean P. falciparum NF54 midgut oocysts in An. stephensi with histamine (hist) provi- Figuresioning 17. inMean the infectedP. falciparum bloodmeal.NF54 midgutN = 4; one oocysts-way ANOVA in An. stephensi (α = 0.05).with * p histamine-value = 0.0152. (hist)provisioning sioning in the infected bloodmeal. N = 4; one-way ANOVA (α = 0.05). * p-value = 0.0152. in the infected bloodmeal. N = 4; one-way ANOVA (α = 0.05). * p-value = 0.0152.

Figure 16. Proportions of An. stephensi infected with P. falciparum NF54 oocysts with histamine (hist) provisioning in the infected bloodmeal. Left: numbers of infected and uninfected mosquitoes by treatment with significant differences noted among pairs. Right panel: data from the left panel shown as percentages of uninfected and infected mosquitoes in each group. N = 4; Chi-square test (α = 0.05). * p = 0.0108.

Biomolecules 2021, 11, 719 Figure 17. Mean P. falciparum NF54 midgut oocysts in An. stephensi with histamine (hist) provi-18 of 23 sioning in the infected bloodmeal. N = 4; one-way ANOVA (α = 0.05). * p-value = 0.0152.

Figure 18. Proportions of An. stephensi with P. falciparum NF54 salivary gland sporozoites with histamine (hist) provision- Figure 18. Proportions of An. stephensi with P. falciparum NF54 salivary gland sporozoites with histamine (hist) provisioning ing in the infected bloodmeal. Left: numbers of mosquitoes with infected and uninfected salivary glands. Right panel: in the infected bloodmeal. Left: numbers of mosquitoes with infected and uninfected salivary glands. Right panel: data data from the left panel shown as percentages of uninfected and infected mosquitoes in each group. N = 1; Chi-square test (fromα = 0.05). the left** p panel= 0.0028, shown **** p as < percentages0.0001. of uninfected and infected mosquitoes in each group. N = 1; Chi-square test (α = 0.05). ** p = 0.0028, **** p < 0.0001. 4. Discussion 4. DiscussionTogether, our data show that histamine ingestion alters the physiology and behavior of uninfectedTogether, and our Plasmodium data show- thatinfected histamine An. stephensi ingestion in altersa manner the physiology that would and be expected behavior of uninfected and Plasmodium-infected An. stephensi in a manner that would be expected to amplify malaria parasite transmission (Figure 19). To support this conclusion, we examined mosquito tendency to take a second bloodmeal, flight activity and investigation of visual objects, neurophysiological responses to odor and light stimuli, lifespan and blood-feeding with increasing age, and infection success with two biologically distinct parasite species. The use of two different concentrations of histamine, including a normal human blood level (1 nM) and a blood level associated with SFM (10 nM) delivered by priming in soaked cotton balls or provisioned in an RBC-serum meal, provided an overview of responses that likely result from histamine detection by An. stephensi midgut receptors, followed by signaling that is transduced via the peripheral and central nervous systems. Both histamine priming and provisioning in blood increased flight activity of CO2- exposed uninfected An. stephensi and histamine priming increased neurophysiological responsiveness to host odors. Host cues, such as odor and visual cues, are sensed by mosquitoes at different distances from the host. CO2 may be detected by mosquitoes at 15–50 m away, whereas visual detection of hosts occurs at much closer distances, at approximately 1–5 m [34]. Histamine is an important neuromodulator and has been shown to selectively impact sensory processing in insects. For example, in certain crepuscular insects, such as Manduca sexta, there is histaminergic coupling between the flight motor system in the thoracic ganglion and the olfactory system in the brain. By contrast, in diurnal insects, like Pieris rapae and D. melanogaster, this coupling occurs between the thoracic ganglion and the visual system [9]. For crepuscular insects that rely on olfactory more than visual cues, this is thought to increase their sensitivity to important odors. Intriguingly, in this study, we found that histamine treatment in An. stephensi increased the proportion of mosquitoes activated by CO2 but did not increase their attraction to visual objects in the wind tunnel at 4 d after treatment. We also found that histamine treatment did not affect retinal (ERG) responses to specific wavelengths and white light. In concert with these findings, we found strong histaminergic staining in the midgut and certain areas of the brain, including strong innervation near the antennal and olfactory lobes. These observations may explain why histamine-treated, uninfected mosquitoes did not exhibit an increased tendency to take a second bloodmeal at 4 d after the first. Additional studies are underway at later time points (i.e., 11 d and 14 d after the first bloodmeal) to fully understand the connections between neurophysiological responsiveness to histamine and these complex behaviors. Biomolecules 2021, 11, x FOR PEER REVIEW 19 of 24

to amplify malaria parasite transmission (Figure 19). To support this conclusion, we examined mosquito tendency to take a second bloodmeal, flight activity and investigation of visual objects, neurophysiological responses to odor and light stimuli, lifespan and blood-feeding with increasing age, and infection success with two biologically distinct parasite species. The use of two different concentrations of histamine, including a normal human blood level (1 nM) and a blood level associated with SFM (10 nM) delivered by priming in soaked cotton balls or provisioned in an RBC-serum meal, provided an over- Biomolecules 2021, 11, 719 view of responses that likely result from histamine detection by An. stephensi midgut19 of re- 23 ceptors, followed by signaling that is transduced via the peripheral and central nervous systems.

FigureFigure 19.19. Ingestion of histamine atat levels associated withwith severesevere malariamalaria cancan enhanceenhance An.An. stephensi behaviors and parasite infectioninfection successsuccess inin aa mannermanner thatthat wouldwould bebe expectedexpected toto amplifyamplify parasiteparasite transmissiontransmission toto andand fromfrom humanhuman hosts.hosts. ((AA)) IfIf aa female mosquito takes a bloodmeal from an uninfected individual (gray host), then subsequently feeds on individuals in female mosquito takes a bloodmeal from an uninfected individual (gray host), then subsequently feeds on individuals in the same population, malaria incidence is not increased. (B) If a female mosquito takes a bloodmeal from an individual the same population, malaria incidence is not increased. (B) If a female mosquito takes a bloodmeal from an individual with severe malaria (blue host), that mosquito may not become infected even if the individual is gametocytemic. However, withingestion severe of malaria elevated (blue blood host), histamine that mosquito (10 nM) may by not this become uninfected infected mosquito even if would the individual be associ isated gametocytemic. with increased However, flight ingestionactivity, responsiveness of elevated blood to histaminehost cues (10and nM) tendency by this uninfectedto take a second mosquito bloodmeal would be from associated this host with population, increased flightproviding activity, an responsivenessincreased opportunity to host cuesfor this and mosquito tendency to to become take a secondinfected bloodmeal (C). Importantly, from this our host observations population, of providing increased an feed increaseding ten- opportunitydency at a more for thisadvanced mosquito age topromoted become infectedby ingested (C). histamine Importantly, could our also observations partially offset of increased age-related feeding declines tendency in blood at a- morefeeding advanced to promote age parasite promoted infection by ingested and transmission. histamine could Once also infected, partially this offset mosquito age-related can transmit declines parasites in blood-feeding to new hosts, to promoteincreasin parasiteg the incidence infection of andmalaria transmission. in the population. Once infected, Mosquitoes this mosquito that become can transmitinfected parasitesafter feeding to new on a hosts, gametocytemic increasing thehost incidence with severe of malaria malaria in and the elevated population. blood Mosquitoes histamine that (D) becomeare more infected likely to after transmit feeding parasites on a gametocytemic to humans because host with an increased proportion carries salivary gland sporozoites. Figure created with BioRender.com. severe malaria and elevated blood histamine (D) are more likely to transmit parasites to humans because an increased proportion carries salivary gland sporozoites. Figure created with BioRender.com. Both histamine priming and provisioning in blood increased flight activity of CO2- exposedWeekly uninfected provisioning An. stephensi of 10 nM and histamine histamine significantly priming increased increased neurophysiological the tendency to blood re- feedsponsiveness from week to host 2 to odo weekrs. 6Host of lifespan cues, such relative as odor to 1and nM visual histamine cues, treatedare sensed mosquitoes. by mos- Longevityquitoes at different and human distances biting from rate have the host. the greatest CO2 may impacts be detected on vectorial by mosquitoes capacity at [50 15],– so50 thism away, persistent whereas response visual to detection histamine of that hosts is independent occurs at much of effects closer over distances, the same at periodapproxi- of timemately on 1 survivorship–5 m [34]. H isistamine striking andis an likely important to amplify neuromodulator parasite infection and andhas transmissionbeen shown byto An.selectively stephensi impact. These sensory observations processing provide in insects. further supportFor example, for our in inferences certain crepuscular that ingested in- histaminesects, such couldas Manduca offset sexta age-related, there isdeclines histaminergic in blood-feeding coupling between by directly the flight increasing motor sys- the tendencytem in the to thora bloodcic feedganglion at advanced and the ages. olfactory system in the brain. By contrast, in diurnal insects,Priming like Pieris with rapae 10 nMand histamineD. melanogaster increased, this coupling the proportion occurs between of An. stephensi the thoracicfemales gan- infectedglion and with the visualP. y. yoelii system17XNL, [9]. For increased crepuscular mean insects midgut that oocysts, rely on and olfactory increased more sporo- than zoite density in the salivary glands relative to controls. Similarly, provisioning of 10 nM histamine in a P. falciparum-infected bloodmeal significantly increased the proportion of mosquitoes with midgut oocysts, increased mean oocysts per midgut, and increased the proportion of females with salivary gland sporozoites. Given that additional blood-feeding has been observed to increase P. falciparum oocyst size and shorten the time to sporozoite appearance in the salivary glands of Anopheles gambiae, changes that could decrease the parasite extrinsic incubation period [51,52], we examined the tendency of P. y. yoelii 17XNL- infected mosquitoes to take a second bloodmeal. Our data suggested that histamine had no effect on the tendency of infected mosquitoes to take a second bloodmeal at 4 d or 11 d PI. Ingestion of histamine during a feeding bout by an uninfected mosquito, however, Biomolecules 2021, 11, 719 20 of 23

on an individual with SFM that did not result in mosquito infection would be predicted to increase the tendency to take a subsequent bloodmeal, and the chances of becoming infected later in adult life. In addition to these factors and in light of the potential for declining levels of salivary gland sporozoites after four weeks [43], increased prevalence and intensity of infection with oocysts and sporozoites could further enhance transmission by older mosquitoes. While we have focused our studies on elevated histamine levels associated with SFM, parasites that are known to co-infect individuals with malaria can also increase circulating blood histamine and increase malarial gametocytemia [53–57]. In this context, anopheline mosquitoes feeding on co-infected patients could exhibit similar changes in physiology and parasite infection success that we have reported here. Co-infections with malaria are clinically, epidemiologically and physiologically complex with outcomes and transmission of the co-infecting malaria parasites driven by the timing of co-infection, host immunity, and the species of co-infecting parasites [53–57]. In this context, it is interesting to speculate that circulating blood histamine levels could function as a predictive biomarker for the risk of malaria parasite transmission in complex clinical co-infections with malaria. In the mammalian host, malaria can trigger a T helper 2 (Th2)-type immune response characterized by elevated blood levels of histamine [2,28,58–61]. In general, a balance between both Th1- and Th2-type immune responses is required for optimal clearance of parasites and to prevent excessive immunopathology due to an overwhelming Th1 response [24,58,61,62]. Importantly, a Th2-biased response has been associated with reduced gametocyte killing and has been shown in animal models to increase parasite transmission to mosquitoes [63–65]. The malaria parasite beneﬁts, therefore, from host manipulation that increases circulating histamine through enhanced survival in the mammalian host and enhanced transmission to mosquitoes. We would suggest that our data demonstrate that enhanced transmission would be extended by direct effects of ingested histamine on mosquito physiology and behavior. Such knowledge could be used to connect clinical interventions (e.g., reducing elevated histamine to mitigate human disease [28]) with the delivery of novel lures [66] to manipulate histamine signaling in vector mosquitoes for improved future malaria control. Accordingly, continued studies of the role of histamine in vector biology will provide novel insights into the complexity of the malaria parasite life cycle.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biom11050719/s1, Figure S1. Plasma histamine in P. y. yoelii 17XNL-infected mice over the course of infection was not increased relative to control, uninfected mice. Author Contributions: Conceptualization, A.M.R., J.A.R. and S.L.; methodology, A.M.R., J.A.R. and S.L.; validation, A.M.R., R.S.-G. and S.L.; formal analysis, A.M.R., E.E.L., J.A.R. and S.L.; investigation, A.M.R., M.G.H., S.J., R.S.-G., C.L. and J.A.R.; resources, J.A.R. and S.L.; data curation, A.M.R. and J.A.R.; writing—original draft preparation, A.M.R. and S.L.; writing—review and editing, A.M.R., E.E.L., J.A.R. and S.L.; visualization, A.M.R., J.A.R. and S.L.; supervision, S.L.; project administration, A.M.R., J.A.R. and S.L.; funding acquisition, J.A.R. and S.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by NIH awards NIAID R01 AI131609 to S.L. and NIAID R21 AI137947 to J.A.R. and by the University of Idaho College of Agricultural and Life Sciences. Institutional Review Board Statement: The study was conducted under approval by the Insti- tutional Animal Care and Use Committee of the University of Idaho (protocol IACUC-2020-10, approved 30 March 2020). Informed Consent Statement: Not applicable. Data Availability Statement: “Wind tunnel data and code are available for download (Riffell, 2021).” Riffell, J.A. Code and data for wind tunnel and electrophysiology recordings. Available online: https://github.com/riffelllab (accessed on 10 May 2021). Biomolecules 2021, 11, 719 21 of 23

Acknowledgments: We thank the Luckhart lab for support for An. stephensi colony maintenance and Jessica Strickland and Sarah M. Garrison of the Luckhart lab for assistance with P. falciparum NF54 culture and for assistance with experimental protocols, respectively. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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