Nutrient acquisition and distribution integrates insect foraging and life history traits.To compensate for deficiencies in specific nutrients at different life stages, insects can obtain these nutrients through supplemental feeding, for example, by feeding on vertebrate secretions in a process known as puddles.The mosquito Anopheles arabiani appears to be malnourished and, therefore, requires nutrients for both metabolism and reproduction.The aim of this study was to assess whether An. arabiensis agitation on cow urine for nutrient acquisition improves life history characteristics.
Make sure it’s safe.arabiensis was attracted to the odor of fresh, 24-hour, 72-hour, and 168-hour old cow urine, and host-seeking and blood-fed (48-hour post-blood meal) females were measured in a Y-tube olfactometer, and pregnant females were assessed for spawning test.A combined chemical and electrophysiological analysis was then used to identify bioactive compounds in cow urine at all four age classes.Synthetic mixtures of bioactive compounds were evaluated in Y-tube and field trials.To investigate cow urine and its main nitrogen-containing compound urea as potential supplemental diets for malaria vectors, feeding parameters and life history characteristics were measured.The proportion of female mosquitoes and the amount of cow urine and urea absorbed were assessed.After feeding, females were assessed for survival, tethered flight, and reproduction.
Seek for the host’s blood and nourishment.In laboratory and field studies, Arabs were drawn to the natural and synthetic scent of fresh and aged cow urine.Pregnant females were indifferent to cow urine responses at spawning sites.Host-seeking and blood-sucking females actively absorb cow urine and urea and allocate these resources according to life-history trade-offs as a function of physiological state for flight, survival, or reproduction.
Anopheles arabinis acquisition and distribution of cow urine for improved life history characteristics.Supplemental feeding of cow urine affects vector capacity directly by increasing daily survival and vector density, and indirectly by altering flight activity and should therefore be considered in future models.
Nutrient acquisition and distribution integrates insect foraging and life history characteristics [1,2,3].Insects are able to select and acquire food and perform compensatory feeding based on food availability and nutrient requirements [1, 3].The distribution of nutrients depends on the life-history process and may lead to different requirements for diet quality and quantity in different life stages of insects [1, 2].To compensate for deficiencies in specific nutrients, insects can obtain these nutrients through supplemental feeding, such as on mud, various excrement and secretions of vertebrates, and carrion, a process known as puddles [2].Although a variety of butterfly and moth species are primarily described, watering holes also occur in other insect orders, and attraction to and feeding on these types of resources can have significant effects on health and other life-history traits [2, 4, 5, 6] ,7].The malaria mosquito Anopheles gambiae sensu lato (sl) emerges as a ‘malnourished’ adult [8], so watering may play an important role in its life history characteristics, but this behavior has so far been neglected .The use of agitation as a means of increasing nutrient intake in this important vehicle warrants attention as this may have important epidemiological consequences.
Nitrogen intake in adult female Anopheles mosquitoes is limited due to low caloric reserves carried from the larval stage and inefficient utilization of blood meal [9].Female Ann.gambiae sl typically compensates for this by supplementing with supplemental blood meals [10, 11], thereby putting more people at risk of contracting the disease and putting mosquitoes at greater risk of predation.Alternatively, mosquitoes can use supplemental feeding of vertebrate excreta to acquire nitrogenous compounds that enhance adaptation and flight maneuverability, as demonstrated by other insects [2].In this regard, the strong and distinct attraction of one of the sibling species within An.The Gambian sl species complex, Anopheles arabinis, fresh and aged cow urine [12,13,14], is interesting.Anopheles arabinis is opportunistic in its host preferences and is known to associate with and feed on cattle.Cow urine is a resource rich in nitrogenous compounds, with urea accounting for 50-95% of the total nitrogen in fresh urine [15, 16].As cow urine ages, microorganisms utilize these resources to reduce the complexity of nitrogenous compounds within 24 hours [15].With the rapid increase in ammonia, associated with a decline in organic nitrogen, alkalophilic microorganisms (many of which produce compounds toxic to mosquitoes) thrive [15], which may be female Ann.arabiensis is preferentially attracted to urine aged 24 hours or less [13, 14].
In this study, host and blood-fed Ans were looked for.During its first gonadotropin cycle, arabiensis was assessed for the acquisition of nitrogenous compounds, including urea, by urine mixing.Next, a series of experiments were conducted to assess how female mosquitoes allocate this potential nutrient resource for improved survival, reproduction and further foraging.Finally, the odor of fresh and aged cow urine was assessed to determine whether these provided reliable clues for host and blood-fed An.In their search for this potential nutritional resource, arabiensis discovered chemical correlations behind the observed differential attractiveness.Synthetic odor mixtures of volatile organic compounds (VOCs) identified in 24-hour aged urine were further evaluated under field conditions, extending the results obtained under laboratory conditions and demonstrating the effect of bovine urine odor on different physiological states. Mosquito attraction.The results obtained confirm that An. arabiensis acquires and distributes nitrogenous compounds found in vertebrate urine to influence life history characteristics.These results are discussed in the context of potential epidemiological consequences and how they can be used for vector surveillance and control.
Anopheles arabicans (Dongola strain) were maintained at 25 ± 2 °C, 65 ± 5% RH and a 12:12 h light:dark cycle.Larvae were reared in plastic trays (20 cm × 18 cm × 7 cm) filled with distilled water and fed Tetramin® fish food (Tetra Werke, Melle, DE).Pupae were collected in 30 ml cups (Nolato Hertila, Åstorp, SE) and then transferred to Bugdorm cages (30 cm × 30 cm × 30 cm; MegaView Science, Taichung, Taiwan) to allow adult emergence.Adults were provided with a 10% sucrose solution ad libitum until 4 days post-emergence (dpe), at which point host-seeking females were offered diet immediately prior to the experiment, or were starved overnight with distilled water prior to the experiment, as described below.Females used for flight tube experiments were starved for only 4-6 hours with water ad libitum.To prepare blood-sucking mosquitoes for subsequent bioassays, 4 dpe females were provided with defibrotic sheep blood (Håtunalab, Bro, SE) using a membrane feeding system (Hemotek Discovery Workshops, Accrington, UK).Fully congested females were then transferred to individual cages and provided diet directly, as described below, or 10% sucrose ad libitum for 3 days prior to the experiments described below.The latter females were used for flight tube bioassays and transferred to the laboratory, and then had distilled water ad libitum for 4-6 hours before the experiment.
Feeding assays were used to quantify urine and urea consumption in adult An.Arab female.Host-seeking and blood-fed females were provided a diet containing 1% diluted fresh and aged cow urine, various concentrations of urea, and two controls (10% sucrose and water) for 48 h.In addition, food coloring (1 mg ml-1 xylene cyanide FF; CAS 2650-17-1; Sigma-Aldrich, Stockholm, SE) was added to the diet and supplied in a 4 × 4 matrix in 250 µl microcentrifuge tubes (Axygen Scientific, Union City, CA, US; Figure 1A) Fill to the edge (~300 µl).To avoid competition between mosquitoes and potential effects of dye color, place 10 mosquitoes in a large Petri dish (12 cm in diameter and 6 cm in height; Semadeni, Ostermundigen, CH; Figure 1A) in complete darkness at 25 ± 2 cm °C and 65 ± 5% relative humidity.These experiments were repeated 5 to 10 times.After exposure to diet, mosquitoes were placed at -20 °C until further analysis.
Look for bovine urine and urea absorbed by the host and blood-sucking female Anopheles arabianus.In feeding trial (A), female mosquitoes were provided with a diet consisting of fresh and aged cow urine, various concentrations of urea, sucrose (10%), and distilled water (H2O).Host-seeking (B) and blood-fed (C) females absorbed more sucrose than any other diet tested.Note that host-seeking females absorbed 72-hour cow urine less than 168-hour cow urine (B).The mean total nitrogen content (± standard deviation) of urine is represented in the inset.Host-seeking (D, F) and blood-sucking (E, G) females take up urea in a dose-dependent manner.Mean inhaled volumes (D, E) with different letter names were significantly different from each other (one-way ANOVA using Tukey’s post hoc analysis; p < 0.05).Error bars represent standard error of the mean (BE).The straight dashed line represents the log-linear regression line (F, G)
To release absorbed food, mosquitoes were individually placed into 1.5 ml microcentrifuge tubes containing 230 µl of distilled water and the tissue was disrupted using a disposable pestle and cordless motor (VWR International, Lund, SE), followed by centrifugation at 10 krpm for 10 min .The supernatant (200 µl) was transferred to a 96-well microplate (Sigma-Aldrich) and absorbance (λ620) was determined using a spectrophotometer-based microplate reader (SPECTROStar® Nano, BMG Labtech, Ortenberg, DE) nm).Alternatively, the mosquitoes were ground in 1 ml of distilled water, 900 µl of which was transferred to a cuvette for spectrophotometric analysis (λ 620 nm; UV 1800, Shimadzu, Kista, SE).To quantify dietary intake, a standard curve was prepared by serial dilution to yield 0.2 µl to 2.4 µl of 1 mg ml-1 xylene cyanide.Then, the optical density of known dye concentrations was used to determine the amount of food each mosquito ingested.
Volume data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc pairwise comparisons (JMP Pro, v14.0.0, SAS Institute Inc., Cary, NC, US, 1989–2007).Linear regression analyses described concentration-dependent urea intake and compared responses between host-seeking and blood-sucking mosquitoes (GraphPad Prism v8.0.0 for Mac, GraphPad Software, San Diego, CA, US).
Approximately 20 µl of urine samples from each age group were bound on Chromosorb® W/AW (10 mg 80/100 mesh, Sigma Aldrich) and encapsulated in tin capsules (8 mm × 5 mm).Capsules were inserted into the combustion chamber of a CHNS/O analyzer (Flash 2000, Thermo Fisher Scientific, Waltham, MA, US) to determine nitrogen content in fresh and aged urine according to the manufacturer’s protocol.Total nitrogen (g N l-1) was quantified based on known urea concentrations used as a standard.
To assess the effect of diet on host-seeking and blood-sucking female survival, mosquitoes were individually placed in large Petri dishes (12 cm in diameter and 6 cm in height; Semadeni) with a mesh-covered hole in the lid (3 cm in diameter) with For ventilation and food supply.Diets were provided directly after 4 dpe and included 1% diluted fresh and aged cow urine, four concentrations of urea, and two controls, 10% sucrose and water.Each diet was pipetted onto a dental tampon (DAB Dental AB, Upplands Väsby, SE) inserted into a 5 ml syringe (Thermo Fisher Scientific, Gothenburg, SE), the plunger removed, and placed on top of a petri dish ( figure 1).1A).Change your diet every day.Maintain the lab as described above.Surviving mosquitoes were counted twice a day, while dead mosquitoes were discarded until the last mosquito died (n = 40 per treatment).Survival of mosquitoes fed on various diets was statistically analyzed using Kaplan-Meyer survival curves and log-rank tests to compare survival distribution comparisons between diets (IBM SPSS Statistics 24.0.0.0).
A custom mosquito flying mill based on Attisano et al.[17], made of 5 mm thick clear acrylic panels (10 cm wide x 10 cm long x 10 cm high) without front and rear panels (Fig. 3: top).A pivot assembly with a vertical tube made of a gas chromatography column (0.25 mm id; 7.5 cm L) with ends glued to an insect needle suspended between a pair of neodymium magnets 9 cm apart.A horizontal tube made of the same material (6.5 cm L) bisected the vertical tube to form a tethered arm and an arm that carried a small piece of aluminum foil as a light-interrupting signal.
24-hour-starved females were given the above diet for 30 minutes prior to restraint.Fully fed female mosquitoes were then individually anesthetized on ice for 2-3 min and attached to insect pins with beeswax (Joel Svenssons Vaxfabrik AB, Munka Ljungby, SE) and then tied to the arms of the horizontal tubes.Flying Mill.Revolutions per flight were recorded by a custom-built data logger, then stored and displayed using PC-Lab 2000™ software (v4.01; Velleman, Gavere, BE).The flight mill was placed in a climate-regulated room (12 h:12 h, light: dark, 25 ± 2 °C, 65 ± 5% RH).
To visualize the pattern of flight activity, the total distance flown (m) and the total number of consecutive flight activities were calculated per hour over a 24-hour period.In addition, the average distances flown by individual females were compared across treatments and analyzed using one-way ANOVA and Tukey’s post hoc analysis (JMP Pro, v14.0.0, SAS Institute Inc.), where average distance was considered a dependent variable , while treatment is an independent factor.Additionally, the average number of rounds is calculated in 10-minute increments.
To assess the effect of diet on reproductive performance of An.arabiensis, six females (4 dpe) were transferred directly to Bugdorm cages (30 cm × 30 cm × 30 cm) after blood collection and then provided the experimental diet for 48 h as described above.Diets were then removed and spawning cups (30 ml; Nolato Hertila) filled with 20 ml of distilled water were provided on the third day for 48 hours, changing every 24 hours.Repeat each dietary regimen 20-50 times.Eggs were counted and recorded for each experimental cage.Subsamples of eggs were used to assess mean size and length variation of individual eggs (n ≥ 200 per diet) using a Dialux-20 microscope (DM1000; Ernst Leitz Wetzlar, Wetzlar, DE) equipped with a Leica Camera (DFC) 320 R2; Leica Microsystems Ltd., DE).The remaining eggs were kept in a climate-controlled room under standard rearing conditions for 24 h, and a subsample of recently emerged 1st instar larvae (n ≥ 200 per diet) were measured, as described above.The number of eggs and the size of eggs and larvae were compared between treatments and using one-way ANOVA and Tukey’s post hoc analysis (JMP Pro, v14.0.0, SAS Institute Inc.).
Headspace volatiles from fresh (1 hr post-sampling), 24 hr, 72 hr and 168 hr aged urine were collected from samples collected from Zebu cattle, Arsi races.For convenience, urine samples were collected early in the morning while the cows were still in the barn.Urine samples were collected from 10 individuals and 100-200 ml of each sample were transferred to individual polyamide baking bags (Toppits Cofresco, Frischhalteprodukte GmbH and Co., Minden, DE) in 3 l polyamide with lid In vinyl chloride plastic drums.Headspace volatiles from each bovine urine sample were collected either directly (fresh) or after maturation at room temperature for 24 h, 72 h and 168 h, ie each urine sample was representative of each age group.
For headspace volatiles collection, a closed-loop system was used to circulate an activated carbon-filtered gas stream (100 ml min-1) through a polyamide bag to the adsorption column for 2.5 h by using a diaphragm vacuum pump (KNF Neuberger, Freiburg, DE).As a control, headspace collection was performed from an empty polyamide bag.The adsorption column was made of Teflon tubing (5.5 cm x 3 mm id) containing 35 mg of Porapak Q (50/80 mesh; Waters Associates, Milford, MA, US) between glass wool plugs.Before use, the column was flushed with 1 ml redistilled n-hexane (Merck, Darmstadt, DE) and 1 ml pentane (99.0% pure solvent GC grade, Sigma Aldrich).The adsorbed volatiles were eluted with 400 μl of pentane.Headspace collections were pooled and then stored at -20°C until used for further analysis.
Behavioral responses of host-seeking and blood-eating An.Headspace volatile extracts collected from fresh, 24-h, 72-h, and 168-h-aged urine were analyzed for volatile extracts from Arabidopsis mosquitoes using a straight glass tube olfactometer [18].The experiments were conducted during ZT 13-15, the peak period of An’s home-seeking activity.Arab [19].A glass tube olfactometer (80 cm × 9.5 cm id) was illuminated with 3 ± 1 lx of red light from above.Charcoal filtered and humidified air flow (25 ± 2 °C, 65 ± 2% relative humidity) passed the bioassay at 30 cm s-1.Air is passed through a series of stainless steel mesh screens, creating a laminar flow and a uniform plume structure.Dental tampon dispenser (4 cm × 1 cm; L:D; DAB Dental AB), suspended from a 5 cm coil on the windward end of the olfactometer, with stimulator changes every 5 minutes.For analysis, 10 μl of each headspace extract, diluted 1:10, was used as a stimulus.An equal amount of pentane was used as a control.Individual host-seeking or blood-sucking mosquitoes were placed in individual release cages 2-3 hours before the start of the experiment.The release cage was placed on the downwind side of the olfactometer, and the mosquitoes were allowed to acclimate for 1 min, and then the butterfly valve of the cage was opened to release.Attraction to treatment or control was analyzed as the proportion of mosquitoes that came into contact with the source within 5 minutes of release.Each headspace volatile extract and control were replicated at least 30 times, and to avoid the effects of any one day, the same number of treatments and controls were tested on each experimental day.Seek responses from host and blood-fed Ans.Arabic versus headspace sets were analyzed using nominal logistic regression followed by pairwise comparisons for odd ratios (JMP Pro, v14.0.0, SAS Institute Inc.).
An’s spawning response.Headspace extracts from fresh and aged cow urine were analyzed in Bugdorm cages (30 cm × 30 cm × 30 cm; MegaView Science).Plastic cups (30 mL; Nolato Hertila) filled with 20 mL of distilled water provided the spawning substrate and were placed in opposite corners of the cage, 24 cm apart.Treatment cups were adjusted with 10 μl of each headspace extract at a 1:10 dilution.An equal amount of pentane was used to adjust the control cup.Treatment and control cups were exchanged between each experiment to control for position effects.Ten blood-fed females were released into experimental cages at ZT 9-11 and eggs in cups were counted 24 hours later.The formula for calculating the spawning index is: (the number of eggs laid in the treatment cup – the number of eggs laid in the control cup)/(the total number of eggs laid).Each treatment was repeated 8 times.
Gas chromatographic and electron antenna pattern detection (GC-EAD) analysis of female An.arabiensis was performed as previously described [20].Briefly, fresh headspace volatile extracts were separated using an Agilent Technologies 6890 GC (Santa Clara, CA, US) equipped with an HP-5 column (30 m × 0.25 mm id, 0.25 μm film thickness, Agilent Technologies). and aging urine.Hydrogen was used as the mobile phase with an average linear flow rate of 45 cm s-1.Each sample (2 μl) was injected for 30 seconds in splitless mode with an inlet temperature of 225 °C.The GC oven temperature was programmed from 35 °C (3 minute hold) to 300 °C (10 minute hold) at 10 °C min-1.In the GC effluent splitter, 4 psi of nitrogen was added and split 1:1 in a Gerstel 3D/2 low dead volume cross (Gerstel, Mülheim, DE) between the flame ionization detector and the EAD.The GC effluent capillary for EAD was passed through a Gerstel ODP-2 transfer line, which tracks the GC oven temperature plus 5 °C, into a glass tube (10 cm × 8 mm), where it was mixed with carbon-filtered, humidified air (1.5 l min−1).The antenna was placed 0.5 cm from the outlet of the tube.Each individual mosquito accounted for one replicate, and for host-seeking mosquitoes, at least three replicates were performed on urine samples of each age.
Identification of bioactive compounds in headspace collections of fresh and aged bovine urine using a combined GC and mass spectrometer (GC-MS; 6890 GC and 5975 MS; Agilent Technologies) to elicit antennal responses in GC-EAD analysis, operating in electron impact ionization mode at 70 eV.The GC was equipped with a HP-5MS UI-coated fused silica capillary column (60 m × 0.25 mm inner diameter, 0.25 μm film thickness) using helium as the mobile phase with an average linear flow rate of 35 cm s-1.A 2 μl sample was injected using the same injector settings and oven temperature as for the GC-EAD analysis.Compounds were identified based on their retention time (Kovát index) and mass spectra compared to the custom library and the NIST14 library (Agilent).Identified compounds were confirmed by injecting authentic standards (Additional File 1: Table S2).For quantification, heptyl acetate (10 ng, 99.8% chemical purity, Aldrich) was injected as an external standard.
Evaluating the efficacy of a synthetic odor mixture consisting of bioactive compounds identified in fresh and aged urine to attract host-seeking and blood-sucking Ans.arabiensis, using the same olfactometer and protocol as above.Synthetic mixtures mimicked the composition and proportions of compounds in mixed headspace volatile extracts of fresh, 24-hour, 48-hour, 72-hour, and 168-hour aged urine (Figure 5D-G; Additional File 1: Table S2).For analysis, use 10 μl of a 1:100 dilution of the fully synthetic mixture, with an overall release rate ranging from approximately 140-2400 ng h-1, for assessing attractiveness to host and blood-sucking mosquitoes.Thereafter, the test is performed on complete mixtures, in which subtractive mixtures of single compounds of the complete mixture are removed.Seek responses from host and blood-fed Ans.Arab vs synthetic and subtractive mixtures were analyzed using nominal logistic regression followed by pairwise comparisons for odd ratios (JMP Pro, v14.0.0, SAS Institute Inc.).
To assess whether cow urine could serve as a host habitat cue for malaria mosquitoes, fresh and aged cow urine, collected as described above, and water were placed in meshed 3 l buckets (100 ml) and set in host bait traps. (BG-HDT version; BioGents, Regensburg, DE).Ten traps placed 50 m apart in pasture, 400 m from village community (Silay, Ethiopia, 5°53´24´´N, 37°29´24´´E) and no cattle, on permanent breeding grounds and villages.Five traps were heated to simulate the presence of a host, while five traps were left unheated.Each treatment location is rotated nightly for a total of five nights.Mosquito numbers captured in traps baited with urine of different ages were compared using logistic regression with a beta binomial distribution (JMP Pro, v14.0.0, SAS Institute Inc.).
In a malaria-endemic village near the town of Maki, Oromia region, Ethiopia (8° 11′ 08″ N, 38° 81′ 70″ E; Figure 6A).The study was conducted between mid-August and mid-September before the annual indoor residual spraying, along with a long rainy season.Five pairs of houses (20–50 m apart) located on the outskirts of the village were selected for the study (Fig. 6A).The criteria used to select the houses were: no animals allowed in the house, no indoor cooking (drawing firewood or charcoal) was allowed (at least during the trial period), and houses with a maximum of two inhabitants, sleeping in uninsecticides. under the treated mosquito net.Ethical approval has been granted by the Institutional Research Ethics Review Board (IRB/022/2016) of the Faculty of Natural Sciences (CNS-IRB), Addis Ababa University, in accordance with the guidelines established by the World Medical Association Declaration of Helsinki.Consent from each head of household was obtained with the assistance of health extension staff.The entire process is endorsed by local administrations at the district and ward (‘kebele’) level.The experimental design followed a 2 × 2 Latin square design, in which synthetic mixtures and controls were assigned to paired houses on the first night and swapped between houses on the next experimental night.This process was repeated ten times.Additionally, to estimate mosquito activity in selected houses, the CDC traps were set to run five consecutive nights at the beginning, middle and end of the field trial at the same time of day.
A synthetic mixture containing six bioactive compounds was dissolved in heptane (97.0% solvent GC grade, Sigma Aldrich) and released at 140 ng h-1 using a cotton wick dispenser [20].The wick dispenser allowed all compounds to be released in constant proportions throughout the 12 hour experiment.Heptane was used as a control.The vial was suspended next to the entry point of the Centers for Disease Control and Prevention (CDC) light trap (John W. Hock Company, Gainesville, FL, US; Figure 6A).The traps were hung 0.8 – 1 m above the ground, near the foot of the bed, and a volunteer slept under an untreated mosquito net and operated between 18:00 and 06:30.Mosquitoes captured by sex and physiological status (unfed, fed, semi-pregnant, and pregnant [21] were subsequently screened using polymerase chain reaction (PCR) analysis to identify the species morphologically identified as A. gambiae sl. Members of the complex [23]. In the field study, trap trapping of paired houses was analyzed using a nominal logistic fit model, where attraction was the dependent variable and treatment (synthetic mixture vs control) was the fixed effect (JMP® 14.0. 0. SAS Institute Inc.). Here, we report the χ2 and p-values ​​from the likelihood ratio test.
Evaluate whether it is safe.arabiensis was able to obtain urine, its main nitrogen source, urea, by direct feeding, within 48 h of administration for 4 days post (dpe) host-seeking and blood-fed female feeding trials (Fig. 1A).Both host-seeking and blood-sucking females absorbed significantly more sucrose than any other diet or water (F(5,426) = 20.15, p < 0.0001 and F(5,299) = 56.00, p < 0.0001, respectively; Fig. 1B,C).Furthermore, host-seeking females ate less in urine at 72 hours compared to urine at 168 hours (Fig. 1B).When offered a diet containing urea, host-seeking females absorbed a significantly greater amount of urea at 2.69 mM compared to all other concentrations and water, while indistinguishable from 10% sucrose (F(10,813) = 15.72, p < 0.0001; Figure 1D).This was in contrast to the response of blood-fed females, who typically absorbed significantly more urea-containing diets than water, albeit significantly less than 10% sucrose (F(10,557) = 78.35, p < 0.0001; Figure 1).1E).Furthermore, when comparing between the two physiological states, phlebotomized females absorbed more urea than host-seeking females at the lowest concentrations, and these females absorbed similar amounts of urea at higher concentrations (F(1,953)= 78.82, p < 0.0001; Fig. 1F, G).While intake from a urea-containing diet appeared to have optimal values ​​(Fig. 1D,E), females in both physiological states were able to modulate the amount of urea absorbed over the entire range of urea concentrations in a log-linear fashion (Fig. 1F,G). ).Similarly, mosquitoes appear to control their nitrogen uptake by regulating the amount of urine absorbed, as the amount of nitrogen in urine is reflected in the amount absorbed (Figure 1B, C and B insets).
To assess the effects of urine and urea on host-seeking and blood-sucking mosquito survival, females were fed urine of all four ages (fresh, 24 h, 72 h, and 168 h post-deposition) and a range of urea concentrations, as well as distilled water and 10 % sucrose served as a control (Figure 2A).This survival analysis showed that diet had a significant effect on overall survival in host-seeking females (urine: χ2 = 108.5, df = 5, p < 0.0001; urea: χ2 = 122.8, df = 5, p < 0.0001; Fig. 2B, C) and blood-fed females (urine: χ2 = 93.0, df = 5, p < 0.0001; urea: χ2 = 137.9, df = 5, p < 0.0001; Figure 2D,E).In all experiments, females fed a diet of urine, urea, and water had significantly lower survival rates compared to females fed a sucrose diet (Figure 2B-E).Host-seeking females fed fresh and stale urine exhibited different survival rates, with those fed 72-h stale urine (p = 0.016) having the lowest survival probability (Fig. 2B).Furthermore, host-seeking females fed 135 mM urea survived longer than water controls (p < 0.04) (Fig. 2C).Compared with water, women fed with fresh urine and 24-hour urine survived longer (p = 0.001 and p = 0.012, respectively; Figure 2D), while women fed with 72-hour urine survived longer than those fed Female short fresh urine and 24-hour aged urine (p < 0.0001 and p = 0.013, respectively; Figure 2D).When fed 135 mM urea, blood-fed females survived longer than all other concentrations of urea and water (p < 0.013; Figure 2E).
Survival of host and blood-sucking female Anopheles arabinis feeding on cow urine and urea.In the bioassay (A), female mosquitoes were provided a diet consisting of fresh and aged cow urine, various concentrations of urea, sucrose (10%) and distilled water (H2O).The survival of host-seeking (B, C) and blood-sucking (D, E) mosquitoes was recorded every 12 hours until all females fed on urine (B, D) and urea (C, E), and controls, Sucrose and water, are dead
The total distance and number of rounds determined in the flight mill test over a 24-hour period differed between host-seeking and blood-sucking mosquitoes, which showed less flight activity overall (Fig. 3).Host-seeking mosquitoes that provided fresh and aged urine or sucrose and water showed distinct flight patterns (Fig. 3), with females feeding on fresh urine being more active at dawn, while those fed 24- and 168-hour aged Mosquitoes that fed on urine exhibited different flight patterns and were primarily diurnal.Female mosquitoes that provided sucrose or 72-hour urine showed activity throughout the 24-hour period, while females that provided water were more active during the mid-period.Mosquitoes fed on sucrose exhibited the highest levels of activity late at night and early in the morning, while those that ingested 72-hour-aged urine experienced a steady decline in activity over 24 hours (Figure 3).
Flight performance of hunter-seeking blood-sucking female Anopheles arabinis feeding on cow urine and urea.In the flight mill test, female mosquitoes fed on fresh and aged cow urine, various concentrations of urea, sucrose (10%), and distilled water (H2O) were tethered to horizontal, freely rotating arms (above).For host-seeking (left) and blood-sucking (right) females, the total distance and number of flights per hour for each diet over a 24-hour period were recorded (dark: grey; light: white).Average distance and average number of bouts are shown to the right of the circadian activity graph.Error bars represent standard error of the mean.Statistical analysis see text
In general, overall flight activity of host-seeking females followed a pattern similar to that of flight distance over a 24-hour period.Mean flight distance was significantly affected by diet ingested (F(5, 138) = 28.27, p < 0.0001), and host-seeking females ingested 72 hours of urine flew significantly longer distances compared to all other diets (p < 0.0001), and sucrose-fed mosquitoes flew longer than fresh (p = 0.022) and 24-h-aged urine (p = 0.022)-fed mosquitoes.In contrast to the flight activity pattern described by the urine diet, urea-fed host-seeking females exhibited persistent flight activity over a 24-h period, peaking during the second half of the dark phase (Fig. 3).Although activity patterns were similar, host-seeking females fed urea significantly increased mean flight distance depending on the absorbed concentration (F(5, 138) = 1310.91, p < 0.0001).Host-seeking females fed any concentration of urea flew longer than females fed either water or sucrose (p < 0.03).
Overall flight activity of blood-sucking mosquitoes was stable and sustained over 24 hours across all diets, with increased urine activity during the second half of the dark period for females fed on water as well as in females fed fresh and 24 hours old (image 3).While urine diet significantly affected mean flight distance in blood-fed females (F(5, 138) = 4.83, p = 0.0004), urea diet did not (F(5, 138) = 1.36, p = 0.24) .with other urine and control diet (fresh, p = 0.0091; 72 hours, p = 0.0022; 168 hours, p = 0.001; sucrose, p = 0.0017; dH2O, p = 0.036).
The effects of urine and urea feeding on reproductive parameters were assessed in egg-laying bioassays (Figure 4A) and were investigated according to the number of eggs laid by each female, egg size, and newly hatched first instar larvae.The number of eggs laid.Urine-fed Arab females varied by diet (F(5,222) = 4.38, p = 0.0008; Fig. 4B).Females fed a 24-hour urine, blood meal laid significantly more eggs than females fed other urine diets and were similar to those fed sucrose (Fig. 4B).Likewise, the size of eggs laid by urine-fed females varied by diet (F(5, 209) = 12.85, p < 0.0001), with 24-hour urine and sucrose-fed females laying significantly larger eggs than water-fed females , while the eggs of females fed with 168 h of urine were significantly smaller (Fig. 4C).In addition, urine diet significantly affected larval size (F(5, 187) = 7.86, p < 0.0001), with significantly larger larvae emerging from eggs laid by 24- and 72-hour-old urine-fed females than from eggs laid from eggs larvae.Water-fed and 168-h urine-fed females (Figure 4D).
Reproductive performance of female Anopheles arabinis feeding on cow urine and urea.Blood-fed female mosquitoes were fed diets consisting of fresh and aged cow urine, various concentrations of urea, sucrose (10%), and distilled water (H2O) for 48 hours before placing in bioassays and obtaining egg-laying substrates48 hours (A).Egg number (B, E), egg size (C, F) and larvae size (D, G) were significantly affected by the diet provided (cow urine: BD; urea: EG).Means for each parameter measured using different letter names were significantly different from each other (one-way ANOVA using Tukey’s post hoc analysis; p < 0.05).Error bars represent standard error of the mean
As the major nitrogenous component of urine, urea, when provided as a diet to blood-fed females, significantly affected reproductive parameters in all studies.The number of eggs laid by females fed urea, after a blood meal, depending on urea concentration (F(11, 360) = 4.69; p < 0.0001), females fed urea concentrations between 134 µM and 1.34 mM laid more eggs ( Figure 4E).Females fed on urea concentrations of 134 µM or above lay larger eggs than females fed on water (F(10, 4245) = 36.7; p < 0.0001; Figure 4F), and larval size, although affected by similar concentrations of urea in mothers (F(10, 3305) = 37.9; p < 0.0001) was more variable (Fig. 4G).
Overall attraction to host-seeking bovine urine headspace volatile extracts.The arabiensis assessed in the glass tube olfactometer (Fig. 5A) was significantly affected by urine age (χ2 = 15.9, df = 4, p = 0.0032; Fig. 5B).Post hoc analysis showed that stale urine odor at 24 hours caused significantly higher levels of attractiveness compared to all other treatments (72 hours: p = 0.0060, 168 hours: p = 0.012, pentane: p = 0.00070), Except for the smell of fresh urine (p = 0.13; Figure 5B).Although the overall attraction of blood-sucking mosquitoes to urine odor was not significantly different (χ2 = 8.78, df = 4, p = 0.067; Fig. 5C), these females were found to be significantly more attractive to headspace volatile extracts compared with 72-hour aged urine compared to controls (p = 0.0066; Figure 5C).
Behavioral responses to natural and synthetic cow urine odors in the search for host and blood-fed Anopheles arabianus.Schematic of the glass tube olfactometer (A).Attraction of headspace volatile extracts from fresh and aged cow urine to host (B) and blood-sucking (C) mosquitoes.Find the tentacle reaction of the Lord An.Headspace extracts isolated from fresh (D), 24-hour (E), 72-hour (F), and 168-hour (G) aged cow urine are shown.Electron antenna detection (EAD) traces show voltage changes in response to bioactive compounds in the headspace eluted from the gas chromatograph and detected by a flame ionization detector (FID).The scale bar represents response amplitude (mV) versus retention time (s).The properties and release rates (µg h-1) of the biologically active compounds are shown.A single asterisk (*) indicates a consistent low-amplitude response.Double asterisks (**) indicate unreproducible responses.Find the host (H) and the blood-sucking (I) An.arabiensis has different appeals to synthetic mixtures of fresh and aged cow urine odors.The mean proportions of mosquitoes attracted to different letter names were significantly different from each other (one-way ANOVA using Tukey’s post hoc analysis; p < 0.05).Error bars represent standard error of the scale
Female Ann.arabiensis, 72 h and 120 h after blood meal, during spawning, no preference was shown for headspace volatile extracts from fresh and aged cow urine compared with pentane controls (χ2 = 3.07, p > 0.05; Additional file 1: Fig. S1).
For female Ann.arabiensis, GC-EAD and GC-MS analyses identified eight, six, three and three bioactive compounds ( Figure 5D-G).Although differences in the number of compounds that elicited electrophysiological responses were observed, most of these compounds were present in each headspace volatile extract collected from fresh and aged urine.Therefore, for each extract, only compounds that produced a physiological response from the female antennae above the threshold were included in further analyses.
The total volatile release rate of bioactive compounds in the headspace collection increased from 29 µg h-1 in fresh urine to 242 µg h-1 in 168-hour aged urine, mainly due to p-cresol and m-formaldehyde Phenol increases as well as phenol.In contrast, the release rates of other compounds, such as 2-cyclohexen-1-one and decanal, decreased with increasing urine age, which correlated with the observed decrease in signal intensity (abundance) in the chromatogram (Fig. 5D)-G left panel) and physiological responses to these compounds (Fig. 5D-G right panel).
Overall, the synthetic mixture had a similar natural ratio of bioactive compounds identified in volatile extracts of fresh and aged urine headspaces (Fig. 5D–G) and did not appear to elicit significant appeal in the search for a host (χ2 = 8.15, df = 4, p = 0.083; Fig. 5H) or blood-sucking mosquitoes (χ2 = 4.91, df = 4, p = 0.30; Fig. 5I).However, post hoc pairwise comparisons between treatments showed that host-seeking mosquitoes were significantly attractive to the synthetic mixture of 24-h aged urine compared with pentane controls (p = 0.0086; Figure 5H).
To assess the role of individual components in synthetic mixtures of 24-h-aged urine, six subtractive mixtures were evaluated against complete mixtures in the Y-tube assay, in which individual compounds were removed.For host-seeking mosquitoes, subtracting individual compounds from the complete mixture had a significant effect on behavioral responses (χ2 = 19.63, df = 6, p = 0.0032; Additional file 1: Figure S2A), all subtractive mixtures were more attractive than Smaller than fully mixed.In contrast, removal of individual compounds from the fully synthetic mixture did not affect the behavioral responses of blood-sucking mosquitoes (χ2 = 11.38, df = 6, p = 0.077), with the exception of decanal, which resulted in lower levels compared to the complete mixture Attraction (p = 0.022; Additional File 1: Figure S2B).
In a malaria-endemic village in Ethiopia, the efficacy of a synthetic mixture of 24-hour cow urine in attracting mosquitoes under field conditions was evaluated for ten nights (Fig. 6A).A total of 4,861 mosquitoes were captured and identified, of which 45.7% were Anthropus.gambiae sl, 18.9% were Anopheles pharoensis and 35.4% were Culex spp.(Additional file 1: Table S1).Anopheles arabinis is the only member of An.Gambian species complex identified by PCR analysis.On average, 320 mosquitoes were captured per night, during which time traps with synthetic mixture baits caught more mosquitoes than paired traps without mixture (χ2(0, 3196) = 170.0, p < 0.0001) .Non-baited traps were set on each of the five control nights at the beginning, middle, and end of the trial.Similar numbers of mosquitoes were captured in each pair of traps, indicating no bias between houses (χ2(0, 1665) = 9 × 10-13, p > 0.05) and no population decline during the study period.Compared with control traps, the number of mosquitoes caught in the traps containing the synthetic mixture was significantly increased: host seeking (χ2(0, 2107) = 138.7, p < 0.0001), recent blood feeding (χ2(0, 650) = 32.2, p < 0.0001) and pregnancy (χ2(0, 228) = 6.27, p = 0.0123; Additional file 1: Table S1).This is also reflected in the total number of mosquitoes captured: host seeking > bloodsucking > pregnant > semi-pregnant > male.
Field evaluation of the efficacy of a 24-hour synthetic cow urine odor mixture.Field trials were conducted in south-central Ethiopia (map), near the town of Maki (insert), using a Centers for Disease Control (CDC) light trap (right) in paired houses, with a Latin square design (aerial image) (A).Synthetic odor-baited CDC phototraps attract and capture female Anopheles arabesques (B), but not Anopheles farroes (C), in a different manner, a physiological state-dependent effect.In addition, these traps captured significantly increased numbers of the host Culex mosquitoes.(D) Compared with control.The bars on the left represent the average selection index of mosquitoes caught in pairs of odorant bait (green) and control (open) traps (N = 10), while the bars on the right represent the average selection index in pairs of control traps (open; N = 5). ).Asterisks indicate statistical significance levels (*p = 0.01 and ***p < 0.0001)
The three species were captured differently in traps containing synthetic mixtures.Looking for host (χ2(1, 1345) = 71.7, p < 0.0001), blood feeding (χ2(1, 517) = 16.7, p < 0.0001) and pregnancy (χ2(1, 180) = 6.11, p = 0.0134) a .arabiensis was trapped in the trap releasing the synthetic mixture (Fig. 6B), while the amount of An did not differ.Pharoensis in different physiological states were found (Fig. 6C).For Culex, only a significant increase in the number of mosquitoes seeking hosts was found in traps baited with the synthetic mixture (χ2(1,1319) = 12.6, p = 0.0004; Fig. 6D), compared with control traps.
Host bait traps located outside of potential hosts between breeding sites and rural communities in Ethiopia were used to assess whether malaria mosquitoes use cow urine odor as a host habitat cue.In the absence of host cues, heat, and with or without the presence of cow urine odor, no mosquitoes were captured (Additional file 1: Figure S3).However, in the presence of high temperature and cow urine odor, female malaria mosquitoes were attracted and captured, albeit in small numbers, independent of urine age (χ2(5, 25) = 2.29, p = 0.13; Additional file 1: Figure S3 ).In contrast, water controls did not capture malaria mosquitoes at high temperatures (Additional File 1: Figure S3).
Malaria mosquitoes acquire and distribute nitrogen-containing compounds through compensatory feeding on cow urine (i.e., puddles) to enhance life-history traits, similar to other insects [2, 4, 24, 25, 26].Cow urine is a readily available renewable resource closely associated with resting places for malaria vectors, such as cowsheds and tall vegetation close to rural homes and spawning sites.Female mosquitoes locate this resource by smell and are able to regulate the uptake of nitrogenous compounds in urine, including urea, the major nitrogenous component in urine [15, 16].Depending on the female mosquito’s physiological state, nutrients in the urine are allocated to enhance the flight activity and survival of host-seeking female mosquitoes, as well as the survival and reproductive characteristics of blood-fed individuals during the first gonadotropic cycle.Therefore, urine mixing plays an important nutritional role for malaria vectors that are closed like malnourished adults [8], as it provides female mosquitoes with the ability to acquire important nitrogenous compounds by engaging in low-risk feeding.This finding has significant epidemiological consequences, as females increase their life expectancy, activity and reproductive output, all of which affect vector capacity.Furthermore, this behavior may be the target of future vector management programs.