The nocturnal swarming activity of the malaria mosquito Anopheles coluzzii: it’s not just the males.

doi:10.21203/rs.3.rs-3124470/v1

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The nocturnal swarming activity of the malaria mosquito Anopheles coluzzii: it’s not just the males.

https://doi.org/10.21203/rs.3.rs-3124470/v1

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Anopheles gambiae sensu lato is the primary malaria vector, endemic to the African continent and has been found to mate in large swarms. To date, the spatial-temporal characterization of mosquito swarms has been restricted to male-focused interactions and relying on visual observations of field events during sunset.

We recorded large-cage laboratory simulated swarm events of Anopheles coluzzii with Photonic Fence Monitoring Device (Photonic Sentry, USA) to characterize various spatial-temporal aspects of male, female, and mixed-sex swarms prior and posterior sunset.

Results suggests that male and female mosquitoes form swarms in a spatially similar manner with no visible structural and physical variations within the tested groups. The study of temporal dynamics of swarms reveals a unique composition of swarm hotspot formation for up to one hour in total during night, which is contrary to literature.

Our results challenge the paradigm that female mosquitoes are acting as reactive individuals to male activity only. We demonstrate that females can form stable and comparable swarms to those of only males or mixed sex, at least under laboratory conditions. This discovery merits further research regarding the purpose of female swarming activity and the missing field observations of females during sunset.

Biological sciences/Biological techniques/Behavioural methods

Biological sciences/Ecology/Behavioural ecology

Anopheles

malaria mosquito

mating

behavior

biomechanics

Mating and swarm formation in the malaria mosquito

Mosquitoes of the Anopheles genus are the primary disease vectors and mate in large complex swarms consisting of several male and female mosquitoes aggregating over a marker structure¹. Anopheles gambiae, Anopheles coluzzii, Anopheles funestus, and Anopheles arabiensis, are the primary disease vectors and endemic to the African continent^2,3 and several of these species have been found to mate in large complex swarms⁴.

Over recent years, a link has been proposed between mating status and Plasmodium transmission whereby females who have mated are more prone to transmit diseases than those who are unmated⁵. Thus, mating mechanisms and sex-specific performance could comprise key information for understanding the malaria disease transmission.

It is throughout the extent of a swarming event that males and females form a copula and fly out of the swarm complex to mate⁶. According to literature, swarm is formed when male mosquitoes aggregate in large numbers over ground markers and typically occurring after sunset⁷. A marker can be constituted of any structure that creates contrast with the ground^8–10.

Male mosquitoes will swarm for up to 20 minutes¹, and females will become attracted to the swarm to find a mating partner¹¹. After approximately twenty seconds, male and female mosquitoes will begin to mate in-flight forming a copula that will leave the swarm to prevent other males to seek mating⁴.

Various studies have given us valuable insight into the general characteristics of swarming behavior, characterizing duration, shape and form, and ecological requirements^6,8,12–16. However, some of the main limitations of these studies involve the methodology followed to gather behavioral information.

The gap in knowledge on male and female swarming activity

The main setback to be encountered when studying the activity of the malaria mosquito is in the observation method. This is because limited visualization possible to be performed with the naked eye when as the daylight decreases.

Few studies have recorded swarms in the field^17–19, and furthermore, observations were not performed for long periods of time. It has been suggested that swarm size can range from 50 to 1000 mosquitoes⁴. There is a limitation to what experimental groups can be studied in the field, with male mosquitoes being the main tested individuals in laboratory and semi-field conditions due to the unpredictability of behavior in field conditions.

On the other hand, studies performed in laboratory conditions may not resemble the natural swarm dynamics found in the field^20,21. This highlights the need for a type of study that can integrate the accurate ecological and behavioral parameters found in the field, with the controlled experimental conditions that can be achieved in a laboratory context.

Based on the aforementioned knowledge, our main goal is to address the gaps of knowledge in the spatial-temporal dynamics of male, female and mixed mosquito swarms by incorporating the use of 3D videography during daylight, sunset and darkness.

Using 3D video tracking for behavioural studies

The use of video tracking software has been previously implemented for characterizing swarming activity in field conditions¹⁹, yet the authors highlight several limitations related to lighting, background, targets, environment, and high volumes of data prove to be quite challenging²². Moreover, post processing linking of trajectory tracks proves to be also demanding based on the erratic nature of mosquito flight²³, especially in swarms.

To challenge this, here we propose to incorporate the use of the Photonic Fence Monitoring Device (Intellectual Ventures Laboratory, Bellevue, WA, USA) for the video tracking of mosquito swarms in laboratory conditions. The main advantage of using this tracking system relies on the user friendliness and capacity to uninterruptedly record moving objects with an accuracy that allows us to reconstruct the two-dimensional and three-dimensional swarm distribution.

Even though swarm composition and physical structure has been previously studied using video image analysis²⁴, filming began only after male mosquitoes were congregating on their site, and recording continued only while males were visibly present. In addition, the exploration of structural parameters of the swarm is mainly male-centric. Although it is known that females can swarm²⁰, their swarming kinematics have not yet been studied systematically. Previous studies on swarming of male Culex pipiens have reported a unique elliptical loop shape characteristic of swarming activity and discuss the ability of male mosquitoes to be able to characterize females based on their wing beat frequency and additionally address that females can form swarms in laboratory conditions²⁰. Yet, the study of temporal components to spatial dynamics was not explored in such detail for long spans of time. This present study provides a foundation for the use of video tracking for behavioral studies of swarming activity in the mosquito Anopheles coluzzii with medical relevance for malaria disease at the African continent.

We postulate that extending the recording time frame from before sunset to after sunset, it may be possible to gather more information on the spatial-temporal dynamics of swarming activity. By carrying out these recordings in laboratory conditions, we can control the sex composition of swarm (experimental groups) and thus expand the study to not only male mosquitoes, but also including the characterization of female-only and mixed-sex swarm dynamics.

Mosquitoes and rearing

We used the BF_Ac strain (F92) of An. coluzzii collected in Vallée Du Kou (Bama, Burkina Faso) in 2014. We reared the colonies in climatic chambers, set at a temperature of 28°C and 80% relative humidity, and providing a 12:12 h light:dark cycle. We fed adult females with horse blood provided with a Hemotek membrane system and hatched the mosquito eggs in water with 0.3% salt. Furthermore, we fed the hatched larvae fed daily using a food slurry preparation consisting of bovine liver powder, tuna meal and Vanderzant vitamin mix²⁵. Afterwards, we sexed individuals at pupal stage under a stereomicroscope, and we let males and females emerge in separate cages (17×17×17 cm) of 100 mosquitoes each. Once adults emerged, we kept the mosquitoes in their cages for a period of five days and provided them with a sugar source (10% sucrose and 2% methylparaben) in each small cage until the beginning of the experiment.

Experimental design

At day five, male and female mosquitoes (in various experimental groups) were released into the large experimental cages (1 x 2.3 x 4 m, W x H x L, 9.2m³) (FIG 1A).

We tested four experimental groups: 100 females, 100 males, 50 females + 50 males (referred to as Mixed 50) and 100 females + 100 males (Mixed 100) which provide an experimental basis that allows the identification of similarities and differences among male and female swarming behavior. Moreover, we additionally included mixed-sex groups to understand the swarm dynamics of interacting groups at low and high densities.

We recorded our experimental groups using a randomized-block design (Fig. 1C) and we performed independent parallel recordings in two large-cage setups. We performed five replicates per experimental group and parallel recordings were randomized over the course of two weeks. For each experimental run, we released either 100 or 200 mosquitoes (depending on the experimental group we tested) and we performed a recording for a period of three hours that covered the activity during one-hour before sunset, sunset and one-hour of night. We performed a total of 20 experimental recordings, using five replicates per experimental group.

To characterize swarm activity, we considered the definition of a swarm by following the approach of two previous studies: in one, a swarm was treated as an up-and-down activity pattern of one or more individuals²⁶. This is complemented by an additional study in which swarming behavior is defined as a collection of mosquitoes flying and forming elliptical loops²⁰.

Large cage setup and protocol.

We performed the swarming experiments our large cage setup consisting of a large cage, an automated light control system, a temperature and humidity control system, a swarming marker, and the Photonic Fence Monitoring Device. Our aim was to use this configuration to understand the collective swarm behavior of our different experimental groups over time, and within a restricted area of interest above the swarm marker.

In addition to the physical set up, we used the visible light system to simulate natural sunset conditions where we provided a daylight, sunset and night-simulated environment by using ceiling led lights of different light temperatures: blue (6500 K), white (4000 K), and yellow (2000 K). The added use of a yellow light in the back of the cage allowed us to simulate sunset light.

The swarm marker was made from a black PVC material and measuring 50 x 50 cm. We this marker at ground level, at ~ 238 cm from the lens of the recording device (FIG 1A, B).

The swarm activity of the four experimental groups was recorded in-flight using the Photonic Fence Monitoring Device (PFMD). For this system to correctly operate, a few parameters were considered: 1) range configuration and 2) retroreflective backstop addition. The range configuration allowed us to determine the desired active region that the camera tracked. Since our cages were 4 m in length, a short-range configuration was used. This provided us a truncated rectangular pyramid range of 2 m base. Moreover, a 3 x 3 m retroreflective backstop was added on the outside of the cage acting as a backlight source. We then initialized the system by pointing towards the desired region of interest above the swarming marker.

System interface and data processing

Once the PFMD camera was set to the desired location and all other cage settings were adjusted, the system was ready to begin recordings. We set the brightness to 100% to visualize moving objects in real-time, in the field of view and over the target. After the recording stopped, we retrieved the tracking data as a zip file containing metadata such as blob log and configurations (virtual interface of Photonic Sentry, https://www.pfmd.net/). The raw data files in CSV format contained temporal information in form of datetime and positional information as X, Y and Z coordinates.

Data selection

Data processing was done using Python 3.8.13²⁷ with the pandas, numpy and matplotlib packages. We estimated the average activity by considering the total number of tracked items per second, as:

N* = N_bin/T_bin, (1)

where N_bin are the number of items detected during a single time bin, and T_bin is the duration of the time bin in seconds. Based on this, a sensitivity analysis was performed using one replicate per experimental group. The decision to perform a sensitivity analysis relay on the ability to discriminate mosquito micromovement e.g., walking on the cage net, versus flight behavior among which swarming activity can be selected. We then calculated the total number of tracked items per unit of time and proceeded to compare between track duration filters of 1 second, 3 seconds, 5 seconds, and 7.5 seconds.

Following time frame selection, all flight tracks were organized based on the individual track ID, thus providing an overview of single path reconstructions within the field of view. Once all tracks have been sorted and grouped, the average flight duration was estimated for each track.

We then proceeded to select a small area above the swarming marker by generating a spatial filter along the z-axis [213, 313 cm] and thus focusing exclusively on mosquito swarming activity above the region of interest as shown in Fig. 1A, B. Furthermore, since the camera system is centered to the middle of the marker, no spatial filter was applied along the x-axis [-50, 50 cm]. A height correction was introduced in each replicate by determining the lowest value and correcting it to fit the Y-axis range of interest [0, 180 cm]. The height of each swarm was measured in relation to its distance to the floor of the cage using the average height (y-coordinate) of all tracked points within an activity period of interest (Fig. 2C, F, I). Two test groups were analyzed based on sex – males versus females - and density – mixed 50 versus mixed 100. The spatial location along the XYZ coordinate axis was characterized as (1) X*: location (in cm) in respect to the middle of the x axis [0,0 cm], (2) Y*: location in respect to height of the y axis [0,180 cm], and (3) Z*: location in respect to the length z axis [213, 313 cm] (Fig. 2).

Statistical analysis

Normality was evaluated for each experimental group utilizing mean data obtained from each replicate. Parameters such as light intensity, temperature, humidity, number of mosquitoes, age, and marker positioning constituted nonrandom factors, whilst swarm shape, height from floor, density, temporal variation, and spatial distribution were considered as random and evaluated for each one of the experimental groups. A Shapiro-Wilk test was computed for each one of the tested factors: X*, Y* and Z* and activity to assess distribution. From the tested factors, only activity showed to be non-normally distributed and thus a Y = sin(Y) transformation was performed to meet normality criteria. A one-way ANOVA with Tukey post-hoc test for multiple comparisons was carried out for each defined activity period (pre-sunset, post sunset and nocturnal activity periods) to compare variation between experimental groups.

A two-way ANOVA test followed by a Tukey post-hoc test with multiple comparisons was performed to analyze the interaction between defined activity period and experimental group. All statistical tests were performed using GraphPad Prism® (Version 9, GraphPad Software Inc., San Diego-CA, USA).

Our definition of a swarm followed the approach of two previous studies: a) a swarm has been characterized by an up-and-down activity pattern of one or more individuals²⁶, b) swarming behavior has been defined as a collection of mosquitoes flying and forming elliptical loops²⁰. Therefore, swarm identification was coupled to characterizing individual point position along the XYZ coordinate axis.

Sensitivity analysis

Preliminary studies on the raw data suggested the need for performing a sensitivity analysis before initiating the post-processing of the flight data. Flight tracks lasting less or equal to 3 seconds were removed from the data frame based on a sensitivity analysis (Suppl. Figure 1, 2 based on an average mosquito speed of 25 cm/s. This speed suggests that a 3 second track duration filter should be the minimum amount of time that is to be considered as a potential participant of a swarm as this is time long enough for a mosquito to go from side to side within the large cages. This allowed us to identify the overall duration of the tracks identified in each recording and thus providing a basis for applying several filtering steps on a reduced dataset.

Moreover, based on the results of the sensitivity analysis, we identified three distinct periods of increased swarming activity (FIG 1D):

A small pre-sunset activity peak that starts thirty minutes before sunset and has a duration of roughly thirty minutes.

A post-sunset activity period that occurs immediately after the cage horizon light is turned off. This period has a duration of twenty minutes and can be seen as a peak of activity common for all experimental groups.

A third increased activity period that after the post-sunset activity peak, called nocturnal activity, and that continues until approximately 60 minutes after sunset.

We used the positional data gathered from each peak to estimate the spatial constitution of swarms in male, female, and mixed groups.

Spatial distribution of mosquito swarms

The relative position relative to the marker for each experimental group and per time period is shown in Table 1. Figure 2 gives us an overview of the average position of the replicates per experimental group. We determined the position of swarms during pre-sunset (Fig. 2A, B, C), post-sunset (Fig. 2D, E, F) and nocturnal activity period (Fig. 2G, H, I) to estimate whether there is a difference in the relative position based on sex and/or density, and not considering time effect in this analysis. We performed a One-Way ANOVA to compare the mean position of X* and Z* of male, female, and mixed groups for each time period separately. Our results suggest that all experimental groups behave similarly in each one of the evaluated activity periods, as no significant differences were found within the mean X* and Z* of the experimental groups. (Pre-sunset activity: X* =-2.097 ± 3.812 m, F = 1.163, P = 0.3716; Z* =287.2 ± 4.134 m, F = 2.912, P = 0.087. Post-sunset activity: X* =1.183 ± 1.474 m, F = 2.457, P = 0.117; Z* =274.6 ± 1.647 m, F = 2.867, P = 0.834. Nocturnal activity period X* =2.355 ± 0.366 m, F = 0.780, P = 0.521; Z* =260.6 ± 1.136 m, F = 0.498, P = 0.688). Moreover, trajectory analysis of each replicate showed that female mosquitoes exhibit the so-called “up and down loops” already throughout the pre-sunset period, unlike males, which exhibit a more distinct loop trajectory during- and after the post-sunset activity period (Suppl. Figure 3, 4).

We additionally evaluated the relative position of the swarms in respect to the y-axis to see whether male, female, and mixed swarms have a comparable height during each independent period of interest. After performing a One-Way ANOVA to compare the mean Y* of the experimental groups, our results indicate all swarm have a similar height at each given activity time of interest, indicating all groups behave similarly when compared independent of the passing of time (pre-sunset activity: height Y* =46.74 ± 6.036 m, F = 3.325, P = 0.064; post-sunset activity: height Y* =64.51 ± 3.225 m, F = 1.378, P = 0.300; nocturnal activity period: height Y* =71.20 ± 0.991 m, F = 0.524, P = 0.671).

Table 1. Summary of swarm spatial parameters across time. The average values per replicate of each experimental group are pooled and summarized to estimate the mean x, y and z position, as well as an overview of the total number of tracked items of each swarm during the (A) pre sunset period, (B) post sunset period, and (C) nocturnal activity period.

Temporal dynamics of mosquito swarming

In addition to the relative spatial position above the marker and height, we estimated the variation in position across the defined times of interest as shown in Fig. 3. By including the temporal component to our analysis, we see a clear moving pattern from all experimental groups towards the center of the marker. As time passes, swarms move away from the back of the cage and towards the left side of the cage, congregating clearly in the middle of the region of interest as seen in Fig. 3A, B. A Two-Way ANOVA was performed on the summary data of each experimental group to evaluate changes in localization over time. A Tukey post hoc analysis of X* indicates female and mixed groups remain along the middle of the marker across the x-coordinate, with no variations in time. Males on the other hand exhibit a left-to-right trajectory when transitioning from pre-sunset to post-sunset activity, which culminates with a centered localization during the nocturnal activity period. It must be noted that looping swarm formation was only visible for males from the post-sunset activity period onwards (Suppl. Figure 3). We also evaluated the z coordinates and saw significant variations of Z* values for each sample time in all experimental group (Fig. 3B, C). A clear approach towards the marker can be seen as time approaches the nocturnal period. Lastly, we estimated whether the height of each experimental group varied across time. Our results show an increasing height pattern for male and mixed groups, yet Y* remains constant for female groups due to the already established swarm formation during the pre-sunset period until nocturnal activity period. Moreover, we see that as time passes swarms tend to go over the 65 cm height mark for all experimental groups (Table 1), showing that female mosquitoes on average exhibit a slightly lower height than male swarms during the post-sunset and nocturnal activity period, which is the first moments in which male swarms can be observed.

Finally, the average activity of each swarm at a given time point was calculated as the total number of tracked items within the pre-sunset, post-sunset, and nocturnal activity periods. The total number of tracked items for each experimental group increasing up to four times once the horizon light was turned off in the set up (Fig. 1C), yet the value of N* remained relatively constant throughout time and withing each experimental group. A Two-Way ANOVA followed by a Tukey post-hoc test was performed indicating no significant differences in the activity of the tested groups (Table 1; Suppl. Figure 5). Given that the first swarm formations become visible during the pre-sunset period of females at a 10231 tracked items (± 11335) per 5 minutes interval, an assumption could be made in terms of activity that a high-density swarm hotspot visualization needs a minimum of 10000 tracked items for an adequate characterization.

Our study elucidates the spatial-temporal characteristics of swarming behavior of males and/or females Anopheles coluzzii. Traditionally, the investigation of the spatial-temporal dynamics and structure of mosquito swarms has been characterized for many years primarily based on analytical techniques that rely on human observation, where past studies provide an initial observational methodology in which insects are visualized and characterized by looking at the lightest part of the sky and waiting until mosquito mating pairs are observed at a naked eye²⁸. One of the main limitations of this type of study is the inability to continue visualizing activity after darkness. By introducing the use of infrared-based three-dimensional video tracking and a three-hour recording timeframe, we were able to capture the nocturnal peak activity of An. coluzzii mosquitoes for more than 1 hour after darkness.

The spatial and temporal characterization of swarming behavior before, during and after sunset revealed that swarming is not a sex-typed behavior. Female mosquitoes can form swarms in a similar structure than male mosquitoes in both, male-only and mixed swarms. Female swarming behavior has previously been explored²¹, however, this study was limited to a communication-based analysis rather than an in-depth structural overview of the hotspot formation over time. The height of female versus male-only swarms does not significantly differ, and in fact, follow a similar trend in terms of their average height above the floor. Such findings suggest the ability of female mosquitoes to group in large numbers even in the absence of a male stimuli. Moreover, the capacity of swarm formation by female mosquitoes raises many more hypotheses on what the initiation cues and/or precursors for it might be.

A field study carried out in Burkina Faso describes the initiation of a swarm by having a male swarm precursor mosquito that flies in a zigzag pattern which results later in the formation of an aggregatory mass of male mosquitoes²⁹. Our results show a comparable extended range of height values during the nocturnal activity period for male-only mosquitoes and a concentrated distribution of values for females. A complex organization of female mosquitoes defies the preconceived knowledge on female attraction to male activity³⁰ and creates a foundation to question this paradigm.

Another theory that could rise from this is a link to this so-called zig zag pattern as to being a behavioral trait of male mosquitoes that continues long after mating copula have been formed in the border zone of the swarm with the purpose of just maintaining the swarm structure and thus acting as a fundamental component for the integrity and maintenance of a swarm complex. The question is raised on whether female mosquitoes also present an “initiation behavior” whenever they aggregate. Here lays one of the limitations of our study in which we are not able to individually and unbiasedly track individual mosquitoes and go more in depth into studying not only swarm formation itself, but also understand where in the swarm are mosquitoes mating and whether the sole purpose of this aggregation is for mating only.

An in-depth literature review was performed into the topic of swarming activity, summarizing the overall knowledge on mosquito swarming behavior across several years and fields of study⁴. Yet, no other behavioral pattern other than mating have been attributed to result from this aggregatory activity. The idea of male mosquitoes forming a swarm complex to which females just approach for mating can thus be challenged by the introduction of these female-only swarms.

From a temporal perspective, we see a series of spatial variations within the experimental groups. We primarily see that as time passes, swarms tend to aggregate towards the center of the swarm marker laid on the floor. This goes in line with reports indicating the exclusive location of swarms over a contrasting structure above the ground⁹. Studies have previously reported a total swarm duration of 12 minutes³¹ however our findings suggest that swarming can take place throughout a two-hour span in the case of females. One explanation for this lies on the limitation of the traditional methodology used in several studies as they rely on visual observation and hence most likely did not identify nocturnal swarming activity. Moreover, in terms of height, we visualize significant differences for all groups across time, except for females. One possibility that could give a reason for female mosquitoes exhibiting condensed behavior is to hypothesize that females display an ordered non-chaotic structure that is not formed as a reactive arrangement to male mating cues.

In other words, females could potentially be able to maintain a clear orderly structure over time, regardless of the presence of males. The main question that rises from our study relates to the inability to visualize female-only swarms in field conditions. One may argue why has a female swarm not yet been observed in the field? And what is the function of female swarming? We also see that during the pre-sunset and post-sunset activity periods, the spread of heights is larger than that of the nocturnal activity period. This could once again suggest a more chaotic behavior previous to the main swarm hotspot formation, shape that has been recently characterized as a ‘swarming-like’ behavior with a unique center core and irregular chaotic behavior on its outside³².

Our study provides a sound basis for the characterization of swarm structure to understand the elemental dynamics of mating and provides evidence of the resembling nocturnal swarming activity found in female mosquitoes in the absence of males or a mating stimulus. Whether this female activity serves also as a mating cue for male attraction needs to be further studied using various sex ratios and incorporating a real-time activity tracking of the spatial-temporal change in swarm structure as a response to the variation of the sex ratios.

Data availability

All raw data generated by the PFMD can be accessed through the Dryad repository: https://doi.org/10.5061/dryad.dfn2z357f.

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The nocturnal swarming activity of the malaria mosquito Anopheles coluzzii: it’s not just the males.

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Abstract

Figures

Introduction

Mating and swarm formation in the malaria mosquito

The gap in knowledge on male and female swarming activity

Using 3D video tracking for behavioural studies

Materials and Methods

Mosquitoes and rearing

Experimental design

System interface and data processing

Data selection

Statistical analysis

Results

Sensitivity analysis

Spatial distribution of mosquito swarms

Temporal dynamics of mosquito swarming

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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