Spatial-Temporal Population Dynamics of Male and Female Aedes Albopictus at a Local Scale in Medellín, Colombia

Background: Diseases transmitted by invasive Aedes aegypti and Aedes albopictus mosquitoes are major public health issues in the tropics and subtropics. Understanding the ecology of mosquito vectors is essential for the development of effective disease mitigation programs and will allow for accurate predictions of vector occurrence and abundance. Studies that examine mosquito population dynamics are typically focused on female presence or total adult captures without discriminating the temporal and spatial distribution of both sexes. Methods: We collected immature and adult mosquitoes twice monthly during a two-year period (2018 – 2019) in the Medellín Botanical Garden (Medellín, Colombia) and assessed: 1) the relationship between climatic variables/vegetation coverage and adult captures, 2) the temporal and spatial distribution of Ae. aegypti and Ae. albopictus during the study period, 3) the temporal and spatial distribution of Ae. albopictus males and females, and 4) the correlation of male and female size in relation to climatic variables and vegetation coverage. Results: We show that Ae. albopictus was the predominant species in the park during the study period. Adult captures were positively correlated with precipitation and relative humidity, and inversely correlated with temperature and wind speed. Spatial analysis showed that Ae. aegypti and Ae. albopictus were distributed at different locations within the surveilled area. Moreover, we observed a spatial misalignment of Ae. albopictus males and females—the majority of males were located in the high vegetation coverage sites and the females in the medium vegetation coverage sites. Conclusions: Our work elucidates the differential dynamics of Ae. albopictus males and females, which is pivotal to develop accurate surveillance and the successful establishment of vector control programs based on the disruption of insect reproduction.

Medellín, Colombia's second largest city, Ae. aegypti was the predominant species until 2011, when the presence of Ae. albopictus was rst detected [19]. Since that time, Ae. albopictus populations have been established in several areas of Medellín (Secretaria de Salud Medellín, unpublished data). Overlapping ecological niches of these two species can result in the competitive displacement of Ae. aegypti by Ae. albopictus or the stabilization and co-existence of both species [20,21].
As Ae. aegypti and Ae. albopictus differ in their human biting behaviors [22] as well as their disease transmission abilities [3], factors that in uence vector abundance can alter disease transmission rates.
Additionally, interactions between Ae. albopictus and Ae. aegypti populations has implications for mosquito control programs that introduce transgenic [23] or Wolbachia-infected [24] Ae. aegypti into the eld. Beginning in 2017 in Medellín, the World Mosquito Program (WMP) began releasing Wolbachiainfected Ae. aegypti males and females to replace native populations [25], as arti cial infection of Ae. aegypti females by Wolbachia blocks transmission of some viruses [26,27]. The continued invasion of Medellín by Ae. albopictus is likely to in uence the successful implementation of this program. Understanding the population dynamics of Ae. albopictus where control programs are implemented may identify factors that in uence the establishment of Wolbachia-infected Ae. aegypti in areas where Ae. albopictus populations exist. Further, elucidation of the spatial dynamics of Aedes males and females will aid control programs that exclusively release males [23,28]. To date, most emphasis has been placed on examining Ae. aegypti population dynamics, but little information regarding Ae. albopictus has been reported.
In the present study, we evaluated the spatial, temporal and population dynamics of male and female Ae. aegypti and Ae. albopictus adults in the Medellín Botanical Garden (Medellín, Antioquia, Colombia), an area with high vegetation and human intervened environments representing several micro-ecosystems, which makes it an excellent open-eld laboratory to test hypotheses on mosquito habitat use and competition. During the study period, Ae. albopictus was the predominant species, accounting for ~ 95% of adult captures. Spatial analysis of captured adults identi ed areas in the Botanical Garden with signi cant associations and disassociations between Ae. aegypti and Ae. albopictus. When we examined Ae. albopictus exclusively, we found that males and females differed in their distributions around the park, and we observed seasonal variation in the distribution patterns of Ae. albopictus males and females. Vegetation coverage had a signi cant in uence on Ae. albopictus captures, with most adults collected in areas with high vegetation. Vegetation coverage also in uenced capture of Ae. albopictus males and females differently: males were primarily captured in areas with high vegetation coverage, while females were evenly distributed in areas with high, medium and low vegetation coverage. Our results show that factors favoring Ae. albopictus populations are present in the Medellín Botanical Garden, and that male and female Ae. albopictus have unique distribution patterns that are at least partially in uenced by vegetation coverage.

Study area
This study was conducted in the Jardín Botánico de Medellín (Medellín Botanical Garden; Fig. 1) that has an area of 14 ha and is located near the city center (6°16 47" W). At 1400 m above sea level, the Medellín Botanical Garden has a subtropical semi-humid climate with vegetation consisting of tropical ora like palms, bromeliads, orchids, ferns, and cycads. The garden is home to various species of birds, small mammals (cats, monkeys and squirrels) and reptiles. The area immediately around the garden consists of residential housing, businesses and other public spaces (a park, a museum and a university), and is adjacent to the city metro line. The Medellín Botanical Garden receives an estimated 130,000 people each month.

Mosquito Collection
Larvae and adult mosquitoes were collected from January 2018 to December 2019 (Fig. 1). Sampling was conducted every two weeks in the late morning (9am-12pm). Adult collections were made at 19 locations within the park (Fig. 1D) employing both human landing catch and sweep nets to capture adults; four researchers remained at each sampling site for 5 min. Captured adults were aspirated into 50 ml conical tubes and labeled with the collection site identi cation code. Larvae were collected at four different locations within the park (Fig. 1D); when present, 20-100ml of water from natural containers (e.g., tree holes) was collected, depending on the volume available. Adult mosquitoes were brought to the laboratory for species and sex identi cation based on morphological characteristics. Wings of adult captures were measured as in [29] to estimate body size. Larvae from each collection site were transferred to 500 ml containers with 100 mL dH 2 O and given a pinch of sh food (Tetramin) until pupation. Pupae were transferred to 5 ml tubes and the species and sex of each specimen was determined upon eclosion.

Spatial analysis ofAedesmosquitoes
We examined differences in adult captures and larvae collected per site per year using a linear mixed model (LMM) with site and year as xed variables, and the month of collection as a random variable in the model. For males and females at each site we used a Generalized linear mixed model (GLMM) with a binomial distribution using each sex over the total captures as the response variable, and site and year as xed variables. Spatial distribution of adult counts was assessed through the Spatial Analysis by Distance Indices (SADIE) methodology [30,31]. SADIE calculates overall aggregation through D, the minimum distance to achieve regularity for the counts in the dataset. The quotient of D and the mean minimum total distance to regularity of thousands of permutations of the dataset yields an overall aggregation index denoted as I a , and a p-value for signi cance with a null hypothesis of randomness (denoted as P a ). When I a > 1, counts are considered aggregated, otherwise this is an indication of regularity. We were interested in detecting patches of consistently high counts relative to the surrounding locations. SADIE provides local indices of clustering, v i for patches and v j for gaps, depending if they are above or below (v i > 1 or v j < -1), or well above or well below the expectation (v i > 1.5 or v j < -1.5), 2 5 " N a n d 7 5 ° 3 3 respectively. These cluster values are then used to calculate neighborhoods of high counts (V i ) or low counts (V j ).
We also tested spatial associations between Ae. albopictus males and females and adult Ae. albopictus and Ae. aegypti using the spatial association test provided by SADIE [32], which tests the signi cance of the association (or disassociation) between two sets of count data, and detects locations where such association is statistically signi cant. The overall spatial association between two datasets, X, is given by the local index x k . If there is presence of either a patch or a gap for the two data, it represents a positive association at the local scale. On the other hand, a negative association, or disassociation, represents a patch for one data and a gap for the other in the same location. Signi cance of X was assessed by comparing the value obtained from the data with the quantiles derived from X rand , the overall index values of 4000 permutations of the two datasets.
Contour maps of local association were constructed by IDW interpolation across the entire sampling region.
Critical values for the contour maps were derived from the quantiles obtained in the permutations. Values of x k that were > 85% of X rand were considered signi cantly associated; those < 85% of X rand were considered signi cantly disassociated. To correct for spatial autocorrelation, critical values were multiplied by an in ation factor derived from the method of [33], after a second-order polynomial detrend [34].

Vegetation coverage analysis
We evaluated the correlation of adult male and female Ae. albopictus captures with the vegetation coverage within the park. We characterized vegetation coverage of the collection sites using the %Cover application (Public Interest Enterprises, Newcastle, Australia); photographs of each collection site were taken 1 m above the ground. Sites were classi ed into ve categories: very high (≥ 90%), high (≥ 80%), medium (≥ 70%), low (≥ 60%) and very low (≥ 40%) ( Table S1). The selection of the sites was intended to represent different ecological settings, ranging from sites near buildings, squares with few plants or trees, and sites with a high density of bushes, plants and trees (sites are described in Table S1). For the statistical analysis, we used the ve established vegetation categories as the xed variable in a LMM, total captures per vegetation classi cation as a random variable, and the number of males, females and total Ae. albopictus as the response variable. Mean comparison was carried out through a Tukey-test.

Temporal analysis ofAedesmosquitoes and correlation with environmental variables
Differences in captures were analyzed per month per year using a LMM, with site as a random factor in the model. Adult and larvae captures were correlated with precipitation, temperature, wind speed, humidity and atmospheric pressure per month. Measurements for these variables during the study period were obtained from the environmental station located in the Botanical Garden maintained by the Sistema de Alerta Temprana de Medellín y el Valle de Aburrá (SIATA; Early alert system of Medellín and the Aburrá Valley). Data from the environmental station is taken each minute; we used monthly averages for the purpose for this study (Table S2).

Wing Size analysis
We used different LMMs to analyze the wing sizes of captured adults. We rst evaluated the overall differences between the sexes and species using each as a xed variable; captures per month per site were used as a random factor in the model. We next analyzed the change in wing sizes using month and year as xed variables and captures per site as a random factor in the model. To analyze wing size distribution within the park we used site and year as xed variables and captures per month as a random factor in the model. We also analyzed the distribution of size across the ve vegetation categories (see above). Finally, we developed a LMM of wing size as a function of each assessed environmental variable assessed (precipitation average, maximum and minimum temperature, wind speed and atmospheric pressure; Table S2).

Aedes albopictusis the predominant species in the Medellín Botanical Garden
From January 2018 -December 2019, adult and premature mosquitoes were collected every two weeks in the Medellín Botanical Garden. At four sites that had consistent water reservoirs (Fig. 1D), we collected 7376 larvae (5591 of which survived to adulthood). Aedes albopictus was the predominant species collected, accounting for 86.1% of the total larvae, followed by Culex spp. (13.9%) and Ae. aegypti (5.8%) ( Table 1). Slightly more male larvae were collected, although both sexes were found in similar proportions (Table 1). Adults were captured at 19 different sites (Fig. 1D); 1398 adults were captured in total. Aedes albopictus was the predominant species (94.6%) followed by Ae. aegypti (4.2%). Culex spp. adults were rarely captured (0.14%). We were unable to identify 1.07% of the adults (Table 1). More adult males (of each Aedes species) were collected overall (Table 1).

Spatial distribution ofAedes aegyptiandAedes albopictusin the Medellín Botanical Garden
Because collection sites were variable (i.e., differences in vegetation, canopy cover, proximity to buildings; Table S1), we examined the spatial distribution of Ae. aegypti and Ae. albopictus within the park and determined exclusion or association sites of both populations. We found signi cant differences in the yearly average of larvae collected at each collection site between 2018 and 2019 (LMM: DF = 3, F = 5.7, p = 0.041; Figure S1A, B). The majority of larvae was collected at bamboo posts and tree holes: sites 9 and 14 (44% and 38% of the total larvae, respectively) in 2018, and at sites 12 and 14 (31.96% and 53.84%) in 2019. Regarding Aedes species, most Ae. aegypti larvae was collected at site 9 (69%) in 2018, and at sites 9 and 14 (39% and 38.5%, respectively) in 2019; most Ae. albopictus larvae was collected at sites 9 and 14 (42.9% and 40%) in 2018, and at sites 12 and 14 (57.8% and 37.6%) in 2019 ( Figure S1A, B).
We next characterized the spatial aggregation of individuals of each species, and the spatial association between Ae. aegypti and Ae. albopictus using SADIE [30,31]. The overall index of aggregation was I a = 1.15 (P a = 0.20) and I a = 1.06 (P a = 0.33) for Ae. aegypti and Ae. Albopictus, respectively, which suggests an overall moderate patchiness of both species across the sampled region that is not signi cantly different from a random pattern. However, we identi ed individual sites where populations aggregate, forming signi cant patches and gaps for each species (Fig. 2C, D). This departure of overall aggregation (I a ) from local indices (v i ) may be a result of small sample sizes or edge effects (i.e., large or small counts consistently around the sampling area)-as in our case-when local indices are more powerful at detecting nonrandom distributions [35,36].
We observed Ae. aegypti aggregations at ve sites and a patch with signi cantly above-average density at site 4 (v i > 1.5; Fig. 2C). There were nine areas with low densities or gaps, while sites 14, 15 and 17 had a signi cantly below-average densities (Fig. 2C). A different local pattern was found for Ae. albopictus: we found aggregations at seven sites with a signi cantly above-average cluster at site 11 (v i > 1.5; Fig. 2D). There were eight sites with low density and a gap at site 17 (v j = -1.5; Fig. 2D). Spatial association between Ae. albopictus and Ae. aegypti was signi cant at sites 4 and 5 (p < 0.05; Fig. 2E), where both species aggregate and the majority of adults were captured ( Fig. 2A, B; Figure S1C, D; Table  S3). Signi cant local disassociations of Ae. aegypti and Ae. albopictus were found at sites 9, 11 and 14 (p < 0.05; Fig. 2E); only Ae. aegypti was aggregated at site 9 and only Ae. albopictus was aggregated at sites 11 and 14. Although we observed co-existence and exclusion locally, we did not observe signi cant disassociation (p = 0.5844) or association (p = 0.415) for the overall population across the study area.

Spatial distribution of male and femaleAedes albopictusin the Medellín Botanical Garden
As we collected more Ae. albopictus males than females during this study (58.4% vs. 41.6%; Table 1), we examined Ae. albopictus male and female distribution within the park to identify areas where they aggregate or disperse. The average proportion of males and females collected at each site did not signi cantly differ between 2018 and 2019 (GLMM: DF = 18, F = 0.654, p = 0.1; Figure S2). However, we observed signi cant differences of the average male-female proportions between sites (GLMM: F = 2.7041, DF = 18, p < 0.001; Fig. 3A, B). The largest proportion of males were collected at sites 4 and 5 ( Fig. 3A; Figure S2), two sites with a high vegetation density (Fig. 1, Table S1). Females had higher proportions at sites 10 and 14 (Fig. 3A, B), sites with low and high vegetation, respectively.
The overall distribution of males and females was moderately patchy, but not signi cantly different from what is expected by chance (Males: I a = 0.9, P a = 0.63; Females I a = 1.18, P a = 0.157). Locally, however, we found that males aggregated in fewer patches than females (Fig. 3C). Additionally, there is a cluster with a high density of females at site 15 (v i > 1.5; Fig. 3D). Both sexes had low densities at seven sites.
However, sites 2, 10 and 19 were occupied by females but not by males. Signi cant gaps for females were found at sites 16 and 17 (v i < -1.5; Fig. 3D). We found that both sexes were signi cantly associated across the sampled area (p = 0.016). Signi cant local associations were observed at sites 5, 7 and 11 ( Fig. 3E), where high numbers of both males and females were recorded (Fig. 3A, B). Site 17 also had a signi cant local association, due to concomitant small counts of both sexes (Fig. 3C, D).
We further analyzed the spatial distribution of males and females during the dry and rainy seasons of Medellín. Medellín has two distinct periods of high and low precipitation annually (see Fig. 5). We combined data for the dry seasons ( rst: January and February; second: June, July and August) and rainy seasons ( rst: March, April and May; second: September, October, and November). At a local scale, we found a similar pattern of patch and gap distribution for both males and females during the rst and second dry season ( Figure S3A, B, E, F). However, the distribution of patches and gaps for both sexes differed between the rst and second wet season. Interestingly, the pattern observed in the second wet season resembled that observed in the rst dry season for both sexes (Figure S3C, D, G, H). Due to the within-year variation observed for the rainy and dry seasons, it is di cult to describe general differences in Ae. albopictus distribution for both sexes. Differences in male and female distribution in the dry and wet seasons may indicate that each sex aggregates differently in space during the year and that this distribution may be in uenced by weather variables. We also observed signi cant associations in certain areas of the park of both sexes, mainly at sites 4 and 5 ( Figure S3I, J, K, L) where high vegetation was found.

Vegetation coverage in uencesAedes albopictuscaptures
We next examined if vegetation coverage in uenced Ae. albopictus captures, classifying each collection site by its percentage vegetation cover, which ranged from very low to very high (Table S1). Vegetation coverage had a signi cant effect on adult captures (LMM: F = 2.8583; DF = 4; p < 0.001; Fig. 4A). The majority of Ae. albopictus were captured at areas classi ed as high or very high vegetation coverage (Fig. 4A). Males and females were uniquely distributed across the differing vegetation coverages (LMM: F = 16.7731; DF = 4; p < 0.001; Fig. 4B)-signi cantly more males were found at areas with very high or high vegetation coverage sites compared to females, who were similarly distributed between sites with low to high vegetation coverage (Fig. 4B).

Aedes albopictushave a bimodal distribution in relation to weather variables
We examined how weather variables correlated with our monthly Ae. albopictus collections. Aedes albopictus captures had a bimodal distribution with distinct peaks during April-May and October-November in both 2018 and 2019, coinciding with months with the highest cumulative precipitation (Fig. 5A); we found a signi cant positive correlation with male and female captures during these months (LMM: p < 0.05; Table S4). Relative humidity also had a signi cant positive correlation with male and female captures (LMM: p < 0.05; Fig. 5B; Table S4). Temperature and wind speed showed a signi cant inverse correlation with adult captures (LMM: p < 0.05; Fig. 6B; Table S4). We also observed a signi cant correlation between precipitation and total larvae collected (LMM: F = 10.19; p = 0.0042; Table S5)-the highest number of larvae collected occurred during months with the highest precipitation in both 2018 and 2019 ( Figure S4), although we also collected larvae in high numbers in January of 2019, a month with low rain levels. We found no signi cant correlation with the total larvae collected for other evaluated environmental variables (Table S5).

Environmental factors in uenceAedes albopictusbody size
Because mosquito body size is related to female longevity and reproductive output [37,38], we examined how Ae. albopictus size changed during the study period and asked if collection site or environmental variables in uenced this trait. Using wing length as a proxy for body size [29], we found signi cant differences in size between species and sex of the adults collected (LMM: DF = 1, F = 4.71, p = 0.030; Figure S5A). Female Ae. albopictus had an average wing length of 2638.16 ± 0.55 µm and males 2197.18 ± 0.31 µm. Female Ae. aegypti had an average wing length of 2914.54 ± 11.26 µm and males 2335.65 ± 7.73 µm ( Figure S5A).
We next analyzed how size changed during the study period by testing the signi cance of month, collection site, environmental variables and size as predictor variables of Ae. albopictus captures. We found that male size signi cantly changed with the month during the study (LMM: DF = 5, F = 2.64, p = 0.022; Figure S5B Figure S5D). We also observed a signi cant association of Ae. albopictus size with precipitation, temperature and wind speed for both sexes (Table S6). Precipitation was directly proportional ( Figure S5E), while temperature and wind speed were both inversely proportional to adult wing size ( Figure S5F, G).

Discussion
Aedes albopictus is a major vector of arboviruses in several countries [39], as the physiological and ecological plasticity of this vector has allowed it to rapidly spread. However, we know little about how climate variables or site characteristics affect the biology and behavior of this species. Further, there is little information regarding how population interactions between Ae. aegypti and Ae. albopictus in uence mosquito behavior. We sampled local mosquito populations for 2 years in the Medellín Botanical Garden (Medellín, Colombia), a high vegetation area surrounded by a dense urban environment.
The Medellín Botanical Garden consists of human intervened environments that are suitable for the establishment of mosquito populations. Aedes albopictus remained the predominant species throughout our study, even considering that the WMP released Wolbachia-infected Ae. aegypti in nearby neighborhoods [40]. We found Ae. aegypti and Ae. albopictus often shared breeding sites but that Ae.
albopictus was always found in higher numbers. The Medellín Botanical Garden is well maintained with little peri-domestic containers present in common areas. However, bromeliads, bamboos, palms, and other plants and trees with the ability to act as breeding sites are commonly found around the park. We collected larva from natural containers-tree holes and bamboo posts-as they were sites that consistently collect water and sustain larvae. Egg-laying preferences may have played a role in Ae. albopictus remaining dominant in the study area, as females prefer to oviposit in natural containers [41] and are attracted to sites with existing larvae [42]. The types of vegetation present in the Botanical Garden may also in uence species predominance, as detritus present in rearing pools can in uence larval competition outcomes, often favoring Ae. albopictus over Ae. aegypti [43]. Aedes albopictus is frequently associated with peri-urban areas with a high density of vegetation coverage, although invasion into urban areas increases larval development rates and adult longevity [44], suggesting that urbanization of Ae. albopictus populations may increase their vectorial capacity. Factors that favor Ae. albopictus establishment may be important in disease transmission by this species, particularly in a dense tropical city such as Medellín.
During our study, more adult males were collected despite using human landing catch and sweep nets to capture adults, the opposite to what has been described using this method [45]. This prompted us to examine the spatial distributions of each sex. While Aedes population density is correlated with increased vegetation [46], we detected unique spatial distributions of males and females that correlated with vegetation coverage. Males were primarily captured in areas with high coverage while females were more evenly distributed. Male and female conspeci cs can have similar spatial distributions based on the homogeneous allocation of resources and/or risks that occur at local scales [47]. However, subpopulation structures based on local resource competition such as breeding sites, mating partners or food sources may occur [48]. For instance, in polygynous species, males have to disperse more widely to nd receptive females, while females have smaller dispersion ranges [48,49]. In monogamous species, however, there may be no bene t for differential dispersion patterns of males and females. Aedes mosquitoes have polygamous males and monogamous females [37,50,51], but we still observed different distribution patterns of males and females, suggesting unique factors may in uence dispersion of Ae. albopictus males and female at a local scale. For instance, male and female feeding preferencesmales feed on nectar while females can feed on nectar and/or blood-feed-may also have contributed to their unique spatial distributions. At sites with high vegetation coverage, we observed signi cant malefemale coexistence and also captured more males than females. These sites had owering plants, suggesting that nectar sources may in uence male-female distributions at local scale, potentially an important consideration for control programs that release only male mosquitoes [23,28].
Climatic variables strongly in uence population dynamics of Ae. aegypti and Ae. albopictus [52]. We found that precipitation was the main environmental factor in uencing mosquito captures. Rainfall was directly proportional to mosquito density-more breeding sites develop to increase the population-and is associated with Ae. albopictus incidence [39]. However, we observed that adult captures were inversely proportional to temperature. Higher temperatures are associated with the Aedes incidence due to optimal conditions for larval rearing with warmer temperatures [39]. However, adult longevity is increased at lower temperatures [53], which may re ect adult activity levels. It is possible that adults are less active with higher temperature, so this negative association might be attributed to our collection methods or the time of day when our collections were conducted.
Body size of adult Ae. albopictus was also in uenced by vegetation and climatic variables. However, the in uence of vegetation on adult size differed by sex. Similar to our adult captures, precipitation was positively correlated with body size and possibly tness, as larger body size is associated with increased fertility in Aedes males and females [37,54,55]. However, adult size decreased as temperature increased. Aedes albopictus females reared at lower temperatures develop into larger adults, with their ovaries displaying higher levels of protein, lipids and carbohydrates than females reared at higher temperatures, which is suggested to contribute to their increased longevity [56]. Interestingly, vegetation coverage in uenced male size, but had no effect on female size. Body size estimations of natural populations, and how environmental factors in uence mosquito size, are important parameters to understand interspeci c competition and can give baseline information for mosquito control programs that are based on insect release.

Conclusions
Our study reveals that Ae. albopictus populations in high vegetation areas such as the Medellín Botanical Garden can remain dominant even if a competitor species is arti cially introduced into surrounding areas. Control methods that release adult mosquitoes into the environment need to consider Ae. albopictus presence and density as a potential complicating factor in their successful establishment. Male and female Ae. albopictus also had unique local distributions, suggesting that local factors in uence how the sexes disperse across the environment. Whether other mosquito species behave similarly, and if similar distributions occur in urban areas with little vegetation, is an area for further exploration. The identi cation of local factors that favor Aedes establishment, and how these factors in uence malefemale distributions, will highlight characteristics that in uence conspeci c population dynamics and ultimately contribute to the success of contemporary mosquito control programs.

Availability of data and material
The datasets generated during and/or analyzed during the current study are available from the corresponding authors upon reasonable request.

Competing interests
The authors declare no competing interests.