Wild Populations of Malaria Vectors Can Mate Both Inside and Outside Human Dwellings

doi:10.21203/rs.3.rs-474590/v1

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Wild Populations of Malaria Vectors Can Mate Both Inside and Outside Human Dwellings

https://doi.org/10.21203/rs.3.rs-474590/v1

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Background: Wild populations of Anopheles mosquitoes are generally thought to mate outdoors in swarms, though once colonized, they also mate readily inside laboratory cages. This study investigated whether malaria vectors, Anopheles funestus and Anopheles arabiensis can naturally mate both inside and outside human dwellings.

Method: Mosquitoes were sampled from three volunteer-occupied experimental huts in a rural Tanzanian village at 6 pm each evening, after which the huts were completely sealed and sampling repeated at 11 pm and 6 am the next morning to compare proportions of inseminated females. Similarly-timed collections were done inside local unsealed village houses. Lastly, wild-caught larvae and pupae were introduced inside or outside experimental huts constructed within two semi-field screen house chambers. The huts were then sealed and fitted with exit traps, allowing mosquito egress but not entry. Mating was assessed in subsequent days by sampling and dissecting emergent adults caught indoors, outdoors and in the exit traps.

Results: Proportions of inseminated females inside the experimental huts in the village increased from ~60% at 6 pm to ~90% the following morning despite no new mosquitoes entering after 6 pm. Insemination in the local homes increased from ~78% to ~93% over the same time points. In the semi-field observations of wild-caught captive mosquitoes, proportions of inseminated An. funestus were 20.9% (95% CI: ± 2.8) outdoors, 25.2% (± 3.4) indoors and 16.8% (± 8.3) in exit traps, while proportions of inseminated An. arabiensis were 42.3% (± 5.5) outdoors, 47.4% (± 4.7) indoors and 37.1% (± 6.8) in exit traps.

Conclusion: This study demonstrates that wild populations of An. funestus and An. arabiensis in Tanzania can mate both inside and outside human dwellings. Most mating likely happens outdoors before the mosquitoes enter houses, but significant additional mating happens indoors. The evidence of insemination inside experimental huts fitted with exit traps and in the unsealed village houses suggests that the mosquitoes can voluntarily mate indoors even when there is unrestricted egress. These findings could enable improved vector control, e.g., by targeting males, and potentially inform alternative methods for colonizing eurygamic Anopheles species (e.g. An. funestus) inside laboratories or semi-field chambers.

Parasitology

mosquito mating

Anopheles funestus

Anopheles arabiensis

Ifakara Health Institute

mosquito swarms

eurygamic species

malaria

Tanzania

Members of the Anopheles gambiae complex and Anopheles funestus group mediate the majority of malaria parasite infections in many parts of sub-Saharan Africa [1–4], where 94% of all malaria deaths and 93% of all cases occurred in 2019 [5]. Countries in this region rely mostly on the use of insecticide-treated nets (ITNs), indoor residual spraying (IRS), as well as effective case management and behaviour change communication to control malaria [6, 7]. These interventions have played a major role in reducing the burden of malaria in the past decades, with vector control interventions contributing to the majority of the success [8–11]. Unfortunately, recent evidence indicates that progress against malaria is plateauing and that gains may be lost [6].

While the major vector control tools, ITNs and IRS primarily target mosquitoes that bite and rest indoors [12–15], it is widely accepted that additional tools targeting other aspects of the mosquito life cycle will be important to further drive progress against malaria [16–18]. One of the opportunities for designing new approaches or optimizing current ones is through an improved understanding of the reproductive behaviours of mosquitoes. For example, a greater understanding of mosquito swarming and mating can enable improved targeting of male mosquitoes [19]. Technologies such as sterile insect techniques (SIT) [20], genetic modification of mosquitoes (GMM) [21], and space spraying (SS) of mosquito swarms [19, 22] are some of the interventions that exploit the mating behaviour of mosquitoes. In Burkina Faso, space spraying of mosquito swarms reduced mosquito populations by up to 80% in the intervention village [19].

Both GMM and SIT have been explored to varying degrees for impact on different insect and pest populations [23–25]. Their successes can be greatly improved if the behaviours of both male and female insects are investigated and exploited. Besides, some of the complementary tools currently being evaluated, notably attractive targeted sugar baits (ATSBs) [26–28], could also be improved by possibly targeting both male and female mosquitoes inside and outside houses.

Like many other diptera, mosquito mating occurs mostly in swarms outside homes [29], where groups of male mosquitoes usually of the same species, aggregate at specific times and places [30]. Several minutes after mosquito swarming begins, females join to seek a mate and leave in copula [22]. Mosquito swarming is thought to be mediated by the circadian rhythm of male mosquitoes [31, 32] and the presence of specific environmental markers in a particular locality [33, 34]. Recent evidence shows that this behaviour might also be mediated by specific aggregation pheromones produced by male mosquitoes [35].

While Anopheles swarms often occur outdoors in open spaces at dusk [36–38], recent collections of resting mosquitoes in south-eastern Tanzania, observed large numbers of male Anopheles resting inside houses at dusk, when the males are supposedly in swarms (Msugupakulya, unpublished). In a separate study, swarms of An. funestus were observed very close to human houses (often at the level of eaves) [36], unlike those of An. arabiensis which tended to occur mostly at the edge of the villages [33]. An experimental hut observation in west Africa found that the daily insemination rates were ~ 5% higher in exit traps than entry traps, implying that some limited mating could occur indoors independent of outdoor swarms [39].

Other than advancing vector control, a greater understanding of Anopheles mating could also improve capabilities for colonizing some of the species that are otherwise difficult to rear inside laboratories. One particular example is An. funestus, which dominates malaria transmission in several east and southern Africa zones [3, 40–43], yet there have been only two successful laboratory colonies using field-collected material from Mozambique and Angola [44]. Recent evidence from attempted colonization has highlighted mating as one of the bottlenecks to colonization [45]. Studying the conditions of successful mating both outdoors in the wild and in captivity could improve rearing of these and other eurygamic mosquito species.

This current study, therefore, investigated indoor and outdoor insemination rates in major malaria vectors (An. funestus and An. arabiensis) under both field and semi-field conditions, to inform new opportunities for improving their control and also to improve efforts to colonize various Anopheles species for laboratory studies.

Study sites

This study was implemented in four phases in the field and semi-field environments. The field studies were done in five villages in Ulanga district, Kivukoni (– 8.2021°S, 36.6961°E), Minepa (– 8.1455°S, 36.4244°E), Tulizamoyo (− 8.3669°S, 36.7336°E), Kilisa (– 8.3721°S, 36.5584°E) and Ruaha (– 8.9068°S, 36.7185°E) and two villages in Kilombero district, Sululu (– 7.9973°S, 36.8317°E) and Ikwambi (– 7.9833°S, 36.8184°E), in south-eastern Tanzania (Fig. 1). The principal malaria vectors in the area are An. arabiensis, and An. funestus [40, 41, 46, 47]. Several other anophelines and some culicine mosquito species are also present in the study area. The semi-field experiments were conducted inside large screen house compartments at the Mosquito City facility maintained by Ifakara Health Institute in Kining’ina village (– 8.1080°S, 36.6668°E) in Kilombero district. These semi-field systems were designed to mimic the natural environment, and have in-build experimental huts for controlled mosquito studies [48].

Study procedures

The four study phases included: (i) assessment of indoor resting densities of male Anopheles mosquitoes relative to females in different house types, (ii) observations of insemination status of wild caught female mosquitoes at different times of night inside volunteer-occupied experimental huts in the village (iii) field observations to assess whether wild mosquitoes’ mate before or after entering local houses occupied by residents and (iv) semi-field observations of wild-caught mosquitoes inside large screen house chambers to verify and quantify the insemination rates inside and outside experimental huts under controlled settings.

Observations of male mosquitoes resting inside different types of houses

Mosquito collections were conducted in 80 houses in the four study villages (Fig. 1), targeting four common house types, namely: (i) 20 houses with thatched-roofs and mud-walls; (ii) 20 houses with thatched-roofs and un-plastered brick-walls; (iii) 20 houses with metal-roofs and un-plastered brick-walls; and (iv) 20 houses with metal-roofs and plastered brick walls. Inside the houses, mosquitoes were collected from various potential resting surfaces using Prokopack aspirators. Initially, mosquito collections were conducted from the morning to noon (6:00 a.m. – 12:00 noon), but additional collections were done in the mornings (7:00 a.m. – 8:30 a.m.), evenings (6:00 p.m. – 8:00 p.m.) and late nights (12:00 a.m. – 2:00 a.m.), as previously described by Msugupakulya et al [49].

Observations of insemination in wild mosquitoes caught inside volunteer-occupied experimental huts

This experiment was conducted in Tulizamoyo village using three volunteer-occupied experimental huts made (Fig. 2). These tent-styled huts consisted of easy-to-seal eave spaces and screened windows, and were located near other village houses used by residents (Fig. 2). During the study, the eave spaces remained open during the day time (7:00 a.m. – 6:00 p.m.) to allow mosquito entrance, and closed at 6 p.m. Trained volunteers entered each experimental hut just before the huts were closed at 6 p.m. and collected mosquitoes using a Prokopack aspirator from multiple indoor surfaces for a total of 5 minutes each time. Without re-opening the huts after the 6 p.m. closure, follow up collections were done at 11 p.m. and again at 6 a.m. the following morning, after which the huts were finally re-opened. At each of these time points, the sampling was done from multiple locations inside the huts. In between the collections, the volunteers slept under untreated nets inside the huts.

It was hypothesized that proportions of inseminated females would stay either similar (if no additional mating happened indoors after 6 p.m. hut closures) or increase (if there was additional mating after 6 p.m.). Collected mosquitoes were kept in labelled cups and transferred to the laboratory for morphological identification and assessment of their insemination status.

The sampled female mosquitoes were firstly identified using morphological keys for Afrotropical Anopheles mosquitoes [50, 51]. An. funestus and An. arabiensis were dissected under a stereo microscope. The seventh segment of immobilized mosquitoes was dissected to extract spermathecae, which was examined under a light microscope with a 10× magnification lens for insemination status. Female mosquitoes with filled long threads coiled brown spermatheca were considered as inseminated while those with clear and non-striated spermathecae were considered as non-inseminated [52]. This study was conducted for 14 consecutive nights in the first round.

After the first round, this experiment was repeated for another ten consecutive nights using the same procedures as described above, except this time all mosquitoes were immobilized by freezing in portable cold-boxes immediately after collection to avoid any possible mating that could happen inside the holding cups.

Observation of insemination in wild female mosquitoes caught from local houses in the study village

An additional experiment to assess the insemination of malaria vectors was performed for five nights using natural houses where people live in the study village. Three thatched-roofed and three iron-roofed houses, all of which were occupied by natives, were used for this experiment. Mosquitoes were collected using the Prokopack aspirators and immobilized immediately in the cooler box with ice packs to prevent any mating potentially happening inside the holding cups and during transportation. These wild-caught mosquitoes were then transported to the insectary and females assessed for their insemination status as described above.

Observations of insemination in wild-caught mosquitoes maintained under semi-field conditions

The semi-field system consisted of large multi-chambered screen houses with netting walls, enclosing village-like ecosystems with vegetation and water puddles [48]. Each chamber (9.6 m x 9.6 m) had an experimental hut constructed to mimic the design of typical local houses used in rural Tanzania (Fig. 3). Three chambers were used for this study. The experimental huts in the selected chambers were completely sealed with mosquito netting on eaves spaces, but the windows were fitted with window-exit traps to catch any mosquitoes attempting to exit the huts. This way, mosquitoes inside the huts could attempt exit (and be trapped in the window exit traps) but those outsides could not enter the huts (since all eave openings were screened, doors closed and windows covered with the exit traps). For additional control, the doors were fitted with overlapping net curtains to prevent mosquitoes from flying in or out whenever someone entered.

Field collection of 3rd and 4th instar Anopheles larvae were done using a combination of standard 350ml dippers (for small habitats) and ten-litre buckets (for large habitats) to maximize densities as previously described by Nambunga et al [53]. The larvae were sorted and only Anopheles used for further observations. The larvae were maintained in rearing basins and fed daily on TetraMin® fish food until they reached the pupae stage.

For each batch of field-collected mosquitoes, the pupae were divided into two approximately equal-sized groups, one of which was put inside the experimental hut in the semi-field chamber, and the second group placed outside the hut. No sex separation of the mosquitoes was done, so each group of pupae yielded both male and female virgins. This experimental set-up was replicated in three different semi-field chambers, each with a similar-sized hut. The field collections were repeated weekly for twelve months, each time adding approximately half of the pupae inside and half outside the huts in each of the three chambers. Emergent adult mosquitoes were recaptured twice weekly from inside and outside the huts (using human landing catches) as well as in the exit traps fitted to the hut windows. The recaptured female mosquitoes were kept in labelled cups and immediately assessed for insemination by dissecting and observing the spermatheca as described above. The observations were done immediately after collections, to minimize the likelihood of mosquitoes mating inside the collection cups.

Molecular identification of An. funestus sibling species

A sub-sample of An. funestus collected from the field and those emerged adults in the semi-field experiments were packed individually in the microcentrifuge tubes with silica gel inside. The samples were transported to the molecular laboratory at Ifakara Health Institute for sibling species identification using polymerase chain reaction (PCR) as described by Koekemoer et al [54].

Statistical analyses

Data were analysed using open-source statistical software, R version 3.6.0 [55]. Proportions and means were used for initial descriptive statistics of mosquitoes in different categories and mean outcomes calculated for each explanatory variable. In the first field survey, generalized linear mixed effect models (glmer), with a negative binomial distribution to account for over-dispersion, were used to assess proportions and variations of male mosquitoes collected inside different house types and at different collection times, i.e., early-morning (7 a.m. – 9 a.m.), late-morning (9 a.m. – 12 p.m.), evening (6 p.m. – 8 p.m.) and midnight (12 a.m. – 2 a.m.). Additionally, pairwise comparisons were performed using Tukey’s Honestly Significant Difference Test using the multcomp package in R.

To compare proportions of inseminated mosquitoes, glmer models with binomial distribution and logit functions were used. For the observations of wild mosquitoes in the village, the mean proportion of inseminated females was modelled as a function of time of mosquito sampling, i.e., evening (6 p.m.), night-time (11 p.m.) and early-morning (6 a.m.). The sampling date and hut identifier were included as random variables in the model. For observations of wild-caught mosquitoes observed in semi-field captivity, the mean proportion of females inseminated was modelled as a function of the location of mosquito recapture, i.e., inside hut, outside hut or in the window exit traps. Again, the sampling date and semi-field chamber identifiers were included as random variables. Results of the models are presented as relative rate ratios (RR) with 95% confidence intervals and their associated p-values.

Observations of male mosquitoes resting inside different types of houses

There were high densities of male mosquitoes collected by Prokopack trap while resting inside all house types included. A total of 27,807 mosquitoes were collected indoors, of which 35.7% (n = 9937) were males. Of these males, 13.1% (n = 1306) were An. funestus, 2.9% (n = 292) were An. arabiensis, 83.9% (n = 8338) were Culex, and only one was An. coustani. House type significantly influenced indoor densities of male mosquitoes. Thatched-roofed houses had slightly higher densities of male Anopheles (but not Culex) mosquitoes than metal-roofed houses (Fig. 4A & Table 1). These indoor densities of Anopheles males were also higher during the early and late morning hours, than in the evening and night time collections (Fig. 4B & Table 1). Overall, the mean number of male An. funestus indoors varied from 0.12 (95% CI: 0.03–0.58) to 0.73 (0.16–3.22), while the mean number of male An. arabiensis varied from 0.03 (0.01–0.11) to 0.20 (0.07–0.59) (Table 2).

Table 1

Summary statistics for pairwise comparison of different mosquito species collected in different house types and different collection times.
	Anopheles funestus		Anopheles arabiensis		Culex mosquitoes
Comparison	Mean difference (95% CI)	p value	Mean difference (95% CI)	p value	Mean difference (95% CI)	p value
House type
Metal roofs and plastered brick walls - Thatched roofs and brick walls	-1.77 (-2.25 - -1.29)	0.001	-1.45 (-2.12 - -0.78)	0.13	-0.07 (-0.46–0.32)	0.998
Metal roofs and un-plastered brick walls - Thatched roofs and brick walls	-0.55 (-1.00 - -0.10)	0.60	-0.22 (-0.78–0.34)	0.98	0.42 (0.04–0.80)	0.68
Thatched roofs and mud walls - Thatched roofs and brick walls	-0.31 (-0.75–0.13)	0.90	0.48 (-0.06–1.02)	0.8	0.29 (-0.10–0.68)	0.87
Metal roofs and un-plastered brick walls - Metal roofs and plastered brick walls	1.21 (0.73–1.69)	0.05	1.23 (0.57–1.89)	0.24	0.49 (0.11–0.87)	0.56
Thatched roofs and mud walls - Metal roofs and plastered brick walls	1.46 (0.99–1.93)	0.01	1.95 (1.31–2.59)	0.01	0.36 (-0.02–0.74)	0.77
Thatched roofs and mud walls - Metal roofs and un-plastered brick walls	0.24 (-0.20–0.68)	0.95	0.7 (0.17–1.23)	0.55	-0.13 (-0.50–0.24)	0.986
Collection time
Evening - Early morning	-0.33 (-0.51 - -0.15)	0.25	-0.69 (-0.99 - -0.39)	0.09	-0.18 (-0.37–0.002)	0.74
Late morning - Early morning	0.09 (-0.15–0.33)	0.98	-0.05 (-0.32–0.22)	0.998	0.11 (-0.06–0.28)	0.90
Mid night - Early morning	-1.69 (-1.90 - -1.48)	< 0.001	-1.47 (-1.82 - -1.12)	< 0.001	-1.29 (-1.48 - -1.10)	< 0.001
Late morning - Evening	0.41 (0.11–0.71)	0.5	0.65 (0.24–1.06)	0.37	0.3 (0.05–0.55)	0.61
Mid night - Evening	-1.36 (-1.58 - -1.14)	< 0.001	-0.77 (-1.18 - -0.36)	0.22	-1.1 (-1.31 - -0.89)	< 0.001
Mid night - Late morning	-1.78 (-2.10 - -1.46)	< 0.001	-1.42 (-1.86 - -0.98)	0.01	-1.4 (-1.65 - -1.15)	< 0.001

Table 2

Showing model estimated means of male *Anopheles* mosquitoes collected from different house types and different collection time points
		Anopheles funestus		Anopheles arabiensis		Culex mosquitoes
	Number of collections	Number of mosquitoes	Mean (95% CI)	Number of mosquitoes	Mean (95% CI)	Number of mosquitoes	Mean (95% CI)
House type
Metal roofs and plastered brick walls	121	54	0.12 (0.03–0.58)	13	0.03 (0.01–0.11)	2385	7.52 (2.16–26.21)
Metal roofs and un-plastered brick walls	134	285	0.42 (0.09–1.87)	35	0.10 (0.04–0.29)	1981	12.34 (3.55–42.90)
Thatched roofs and brick walls	128	428	0.73 (0.16–3.220	58	0.13 (0.04–0.36)	1614	8.08 (2.33–27.97)
Thatched roofs and mud walls	143	539	0.53 (0.12–2.41)	186	0.20 (0.07–0.59)	2358	10.84 (3.10-37.92)
Collection time
Early-morning	273	851	0.36 (0.08–1.49)	136	0.10 (0.04–0.26)	5101	9.47 (3.09–29.07)
Late-morning	87	142	0.39 (0.09–1.66)	125	0.09 (0.03–0.27)	2278	10.62 (3.38–33.36)
Evening	78	248	0.26 (0.06–1.11)	18	0.05 (0.02–0.15)	671	7.89 (2.44–25.48)
Mid night	86	65	0.07 (0.01–0.29)	10	0.02 (0.01–0.10)	239	2.61 (0.80–8.49)
Means were calculated from glmer models

Lastly, PCR analysis showed that 93% of the adult An. funestus mosquitoes caught indoors in these villages are An. funestus s.s, the rest being An. rivulorum, and 100% of adult An. gambiae complex mosquitoes were An. arabiensis.

Observations of insemination in wild mosquitoes caught inside volunteer-occupied tented huts in rural Tanzania

The proportion of inseminated females increased significantly after the 6 p.m. collections and was highest in the morning collections done at 6 a.m., even though the huts remained completely sealed from 6 p.m. A total of 594 An. funestus (489 females and 105 males) and 795 An. arabiensis (647 female and 148 male) were collected over the first 14 nights. The mean proportion of inseminated An. funestus mosquitoes increased from 60.7% (95% CI: ± 7.2%) at the 6 p.m. collections to 79.6% (± 5.1%) at 11 p.m. and 92.8% (± 3.8%) the following morning at 6 a.m. Similarly, mean proportion of inseminated An. arabiensis females increased from 60.6% (± 4.8%) at 6 p.m. to 80.4% (± 4.1%) at 11 p.m. and 88.1% (± 4.4%) the following morning at 6 a.m. (Fig. 5A & Table 3). The glmer analysis showed that the increases were statistically significant (p < 0.001) (Table 3).

Table 3

Mean proportion of insemination of *Anopheles funestus* and *An. arabiensis* collected at different time points
		Anopheles funestus				Anopheles arabiensis
		No. females collected	Proportion inseminated (95%CI)	OR (95%CI) for insemination	p value	No. females collected	Proportion inseminated (95%CI)	OR (95%CI) for insemination	p value
Round 1 Studies in Experimental Huts in rural Tanzania (14 nights)	Evening (6:00 p.m.)	191	60.7% (± 7.2)	Ref		228	60.6% (± 4.8)	Ref
	Night (11:00 p.m.)	175	79.6% (± 5.1)	2.39 (1.50–3.79)	< 0.001	240	80.4% (± 4.1)	2.54 (1.69–3.83)	< 0.001
	Morning (6:00 a.m.)	123	92.8% (± 3.8)	6.10 (3.14–11.85)	< 0.001	179	88.1% (± 4.4)	4.91 (2.90–8.31)	< 0.001
Round 2 Studies in Experimental Huts in rural Tanzania (10 nights)	Evening (6:00 p.m.)	17	25% (± 19.8)	Ref		37	46.1% (± 8)	Ref
	Night (11:00 p.m.)	12	71.4% (± 29.1)	2.28 (1.14–3.51)	0.071	36	72.4% (± 17.2)	2.46 (1.36–4.58)	0.046
	Morning (6:00 a.m.)	16	83.3% (± 16.3)	3.12 (1.26–5.42)	< 0.001	36	74.6% (± 12.5)	4.42 (2.02–6.21)	< 0.001
Studies in local resident-occupied village houses in rural Tanzania	Evening (6:00 p.m.)	130	75.5% (± 7.7)	Ref		115	80.9% (± 8.1)	Ref
	Night (11:00 p.m.)	107	85.9% (± 7.1)	2.09 (1.06–4.14)	0.03	128	87.6% (± 5.9)	1.63 (0.83–3.19)	0.15
	Morning (6:00 a.m.)	113	94.7% (± 4.0)	6.14 (2.56–14.74)	< 0.001	126	91.2% (± 4.7)	2.79 (1.30–6.02)	0.01

Analysis of data from the second round of the experiment (conducted over 10 nights, during which the captured mosquitoes were immediately immobilized) revealed a similar trend of increasing proportion of insemination from 6 p.m. to 6 a.m. The mean proportion of inseminated An. funestus females increased from 25% (95% CI: ± 19.8%) at 6 p.m. to 71.4% (± 29.1%) at 11 p.m. and 83.3% (± 16.3%) at 6 a.m. On the other hand, the mean proportion of An. arabiensis increased from 46.1% (± 8%) at 6 p.m. to 72.4% (± 17.2%) at 11 p.m. and finally 74.6% (± 12.5%) at 6 a.m. (Fig. 5B and Table 3).

The male to female ratio for An. funestus and An. arabiensis caught in the experimental huts at the different collection times are also shown (Fig. 6). PCR analysis showed that of all An. funestus collected from the field experiment, 94% were An. funestus s.s with the other 6% unamplified, while An. arabiensis was the only member of An. gambiae complex in the area.

Observation of insemination in the wild mosquitoes caught from local houses occupied by natives in the study village

Similar to wild mosquitoes caught inside volunteer-occupied experimental huts, the proportion of inseminated females caught from houses in the study villages increased significantly after the 6 p.m. collections, and was highest in the morning collections. A total of 350 female and 64 male An. funestus; and 369 female and 74 male An. arabiensis were collected over five nights of this experiment.

Regardless of house type, the mean proportion of inseminated An. funestus and An. arabiensis females increased overnight (Fig. 7). The mean proportion of An. funestus increased from 76.4% (95% CI: (± 9.9%) at 6 p.m. to 88.8% (± 9.9%) at 11 p.m. to 93.3% (± 6.2%) at 6 a.m. in metal-roofed houses; and from 74.7% (± 12.1%) at 6 p.m. to 82.7% (± 10.3%) at 11 p.m. to 96.1% (± 5.3%) at 6 a.m. in thatched roof houses. For An. arabiensis, the mean proportion of insemination increased from 81.8% (± 10.9%) at 6 p.m. to 89.2% (± 6.0%) at 11 p.m. to 90.8% (± 6.2%) at 6 a.m. in metal-roofed and 80% (± 12.5%) at 6 p.m. to 89.4% (± 9.2%) at 11 p.m. to 91.5% (± 7.4%) at 6 a.m. in thatched roofed houses (Fig. 7).

In a combined analysis of the house types, a significant increase in the proportion of inseminated female Anopheles mosquitoes was observed from evening to morning. However, with An. arabiensis, the difference in the proportion of inseminated mosquitoes collected between 6 p.m. and 11 p.m. was marginal (Table 3).

Observations of insemination in wild-caught Anopheles mosquitoes maintained under semi-field conditions

In the semi-field compartments where wild-caught mosquitoes were held captive either inside or outside experimental huts (Fig. 3), a total of 3,241 female Anopheles mosquitoes were recaptured following multiple collections over the twelve months experiment duration. Of these, 56.6% (n = 1833) were An. funestus and 43.4% (n = 1408) were An. arabiensis. Overall, 25.5% of An. funestus and 45.5% of An. arabiensis were caught inseminated.

The mean proportion of inseminated An. funestus females was 16.8% (± 8.3%) in the exit traps, 25.2% (± 3.4%) inside the huts and 20.9% (± 2.8%) outside the huts. On the other hand, mean proportions of inseminated An. arabiensis females was 37.1% (± 6.8%) in the window exit traps, 47.4% (± 4.7%) inside and 42.3% (± 5.5%) outside the huts (Table 4). Further analysis of the An. funestus data showed that the insemination rates were significantly higher indoors and lower in the exit traps compared to the outdoors (p ≤ 0.03). There was also a difference in insemination rates in An. arabiensis caught from indoors compared to outdoors though this was not statistically significant (p = 0.11). The same was observed for this species in samples collected from exit traps and outdoors, for which their difference was marginal (p = 0.13) (Table 4).

Table 4

Mean proportion of inseminated *Anopheles funestus* and *Anopheles arabiensis* from semi-field experiment
		Anopheles funestus				Anopheles arabiensis
		No. females collected	Proportion inseminated (95%CI)	OR (95%CI) for insemination	p value	No. females collected	Proportion inseminated (95%CI)	OR (95%CI) for insemination	p value
Studies in semi-field system	Outdoor	908	20.9% (± 2.8)	Ref		568	42.3% (± 5.5)	Ref
	Indoor	792	25.2% (± 3.4)	0.56 (0.51–0.62)	0.03	568	47.4% (± 4.7)	0.55 (0.49–0.61)	0.11
	Exit trap	133	16.8% (± 8.3)	0.36 (0.26–0.48)	0.026	298	37.1% (± 6.8)	0.44 (0.37–0.52)	0.13
Interspecies comparison in a semi-field experiment		Indoor		Outdoor		Exit trap
		OR (95%CI)	p value	OR (95%CI)	p value	OR (95%CI)		p value
	An. funestus	Ref		Ref		Ref
	An. arabiensis	1.79 (1.33–2.41)	< 0.001	2.23 (1.62–3.08)	< 0.001	2.94 (1.77–4.90)		< 0.001

Proportions of inseminated An. arabiensis were consistently higher than proportions of inseminated An. funestus across all collection locations (outdoors, indoors as well as in the window exit traps) (p < 0.001) (Table 4).

After PCR analysis of the emerged adult An. funestus in the semi-field study, 84.9% were An. funestus s.s, 6.5% were Anopheles rivulorum and 0.9% were Anopheles leesoni. While most of the mosquitoes mating indoors and outdoors were An. funestus s.s. or An. arabiensis, there were also An. rivulorum mosquitoes mated both indoors and outdoors.

Like many other Dipterans, mosquito mating is considered to occur mostly in swarms in different arenas [30], but it is recognized that mating can also occur independently of swarms [39]. The focus of this study was to investigate the indoor and outdoor mating behaviours of the main malaria vectors, An. funestus and An. arabiensis in rural Tanzania. The study used a 4-phase approach consisting of: (i) assessment of indoor resting densities of male mosquitoes inside human dwellings in rural villages, (ii) field observations of wild mosquitoes entering volunteer-occupied huts constructed in the same villages (iii) field observation of insemination status of wild mosquitoes trapped from natural houses in the village, and (iv) controlled semi-field observations of wild-caught mosquitoes to verify and quantify insemination indoors and outdoors.

The three main findings were as follows: (i) significant proportions of male mosquitoes rest indoors alongside females and the densities of these males are highest during in the mornings, (ii) approximately 60% of female Anopheles mosquitoes collected indoors were already inseminated at 6 p.m. but this proportion increased to 90% by the following morning even when the huts remained closed after the 6.p.m collection, implying additional mating indoors despite no additional mosquito-entry, and (iii) under controlled semi-field settings, wild-caught Anopheles mosquitoes held inside huts mated as much as those outdoors.

These observations confirm that while most mating in the wild may be happening outdoors, there is substantial additional mating that can happen after the mosquitoes are already indoors. Both An. funestus and An. arabiensis express this flexibility in behaviours though indoor insemination appeared more prominent in Anopheles funestus, for which indoor insemination exceeded that outdoors. The experimental hut observations done by Dao et al in west Africa observed 5% higher mating in mosquitoes exiting huts than those entering, thus providing the first indications of this phenomenon in malaria mosquitoes [39]. This new study supports that original hypothesis but also demonstrates that indoor mating under some conditions can be far more substantial. Moreover, unlike the outdoor mating in swarms, which typically happen at dusk [33, 36, 56, 57], this study shows that indoor mating can happen far later in the evenings and during the night. The field study showed a gradual increase in insemination from approximately 60% at 6 p.m. to 80% at 11 p.m. and 90% at 6 a.m. the following morning, suggesting continued mating events throughout the night. Since no observations were made of any swarms indoors, it is unclear whether the indoor mating is a function of swarms or otherwise.

This study also highlights the influence of different house types on indoor-resting densities of male mosquitoes. There was preference of males of both An. funestus and An. arabiensis for less improved houses such as houses with thatched roofs compared to more improved houses such as those with metal roofs. Similar preferences have are already well-demonstrated in females Anopheles in several studies [49, 58, 59]. One clear implication for this is that indoor interventions such as indoor residual spraying (IRS) [15], which typically target host-seeking females may also be impactful on the males. This may explain why IRS campaigns have been particularly effective against An. funestus populations, for which substantial densities of males rest indoors [60, 61]. Fortunately, insecticide-resistance surveys in rural Tanzania have shown that male Anopheles have a similar phenotypic expression of resistance as their female conspecifics, and would therefore respond similarly to non-pyrethroid IRS treatments [62]. Similarly, some of the new vector control tools being evaluated to complement ITNs and IRS, notably attractive toxic sugar baits [26, 28, 63] could be optimized to target male and female mosquitoes indoors.

An important question is whether the experimental set-up used in this study induced the observed indoor insemination or if this was entirely a natural phenomenon. While it is impossible to rule this out, the actual findings in totality suggest that indoor mating is a natural phenomenon, albeit occurring at a lower frequency than outdoor mating. The natural observation of high densities of males indoors indicates significant opportunities for copulation between sexes. Moreover, the experimental huts used in the semi-field observations were fitted with window exit traps so that flying mosquitoes could exit but not enter the huts. Yet there was still substantial insemination indoors, the mean proportions being equal to or higher than those observed outdoors or in exit traps. It can be inferred therefore that both An. funestus and An. arabiensis adults could voluntarily mate indoors despite the unrestricted egress routes. This was confirmed by the observed indoor insemination in local village houses, which remained unsealed during the experiment.

To accelerate efforts towards malaria elimination, the deployment of novel approaches to complement current vector control (ITNs and IRS) are essential [16]. Such tools should particularly mitigate current challenges such as insecticide resistance to the commonly-used pyrethroids [64–67] and outdoor-biting by malaria vectors [68–73]. The findings of this study mating behaviour, among other aspects of Anopheles life cycle processes may provide insights towards the development or improvement of malaria vector control. The evidence may be used to design or improve indoor insecticidal methods targeting males, females or both, as well as niche products such as ATSBs to also target male and female mosquitoes indoors and outdoors.

Another potential opportunity from these findings is in the area of mosquito colonization inside laboratories, which is currently a challenge for some vector species. In particular, An. funestus, which mediates significant proportions of ongoing malaria transmission, particularly in southern and eastern Africa, remains one of the most difficult to rear inside laboratories. Laboratory colonies allow experimental studies under controlled conditions, e.g. enabling characterization of insecticide resistance [62, 74, 75] as well as studies on other genetic traits [76, 77]), immune strategies [78, 79] and key vector demographic profiles [80–82]. Unlike An. gambiae, An. funestus remains extremely difficult to colonize and maintain inside laboratories, partly because it is eurygamic (does not mate in captivity) and has poorly understood ecological needs. To our knowledge, only two strains have been successfully colonized from wild populations despite several attempts, both at the Vector Control Reference Laboratory (VCRL) in the National Institute for Communicable Diseases, South Africa, from populations in Angola (FANG) and Mozambique (FUMOZ) [44]. The FUMOZ strain also maintained at other laboratories worldwide including in Cameroon, UK [75] and Tanzania [45].

Several attempts are being made to colonize new An. funestus strains [83] from wild populations, but methods used to establish FUMOZ and FANG have not been successful elsewhere [84], including when attempted in the same wild populations where FUMOZ was originally derived (Coetzee personal communication). The inability to repeatedly colonize and establish An. funestus in laboratories is responsible for the more limited understanding of the biology of this species compared to other vector species. Recent evidence from attempted colonization in Tanzania has highlighted mating as one of the bottlenecks to colonization [45]. The findings in this current study may therefore enable advancement towards alternative colonization processes either inside laboratories or by using semi-field chambers and experimental huts in which natural mating can happen.

This study demonstrates that wild populations of An. funestus and An. arabiensis can mate both inside and outside dwellings. Most mating likely happens outdoors before the mosquitoes enter houses, but significant additional mating can happen indoors. The indoor insemination in huts with exit traps indicates that mosquitoes can voluntarily mate indoors despite unrestricted egress. These findings may be relevant for improving vector control by targeting male mosquitoes and may also inform improving efforts to colonize Anopheles species inside laboratories or semi-field chambers.

ITNs: Insecticide-treated nets; IRS: Indoor Residual Spraying; SIT: Sterile Insect Techniques; CI: Confidence intervals; GMM: Genetically modified mosquitoes; SS: Space spraying; PCR: Polymerase chain reaction

Author’s contributions

IHN, BJM, MH, EWK, HSN and FOO conceived the study and developed the study protocol. IHN, and BJM produced the first drafts of the manuscript. IHN, BJM, HSN, LLM and FOO analysed the data. IHN, BJM, IHM, NFK and EWK conducted the experiments. IHN, GM and DMM dissected the mosquitoes. IHN, BJM and NFK created the study site map. IHN, BJM, HSN, LLM, IHM, NFK, RMN, EEH, MH, FT, HMF, EWK and FOO reviewed the manuscript and provided inputs. All authors have read and contributed to the final manuscript.

Acknowledgements

Many thanks to the volunteers who participated in the collection of mosquitoes for both field and semi-field experiments. Many thanks should go to Gerald Tamayamali, Nicolaus Mhumbira, Richard Lumuli and Karimu Mchwembo for assisting in larval collection for the semi-field experiment. Also, the authors would like to provide great thanks to all staffs at Ifakara Health Institute for providing great supports and opinions regarding the experiment.

Conflict of interest

The authors declared that they have no competing interests.

Availability of data and materials

All data generated from this study will be available from the corresponding authors upon request.

Funding

The activities in this work were supported by Howard Hughes Medical Institute (HHMI) – Gates International Research Scholar Award to FO at Ifakara Health Institute (OPP1175877) and Bill & Melinda Gates Foundation Grant, also to Ifakara Health Institute (INV-002138). IHN was funded by the University of Glasgow GCRF Small Grants Fund, which is supported by a Global Challenges Research Fund allocation from the Scottish Funding Council. Also, EWK was funded by NIHR–Wellcome Trust Partnership for Global Health Research International Training Fellowship (Grant Number: 216448/Z/19/Z).

Ethics approval and consent to participate

Ethical approval for this study was obtained from Ifakara Health Institute Institutional Review Board (IHI/IRB/No: 26-2020), and the National Institute of Medical Research (NIMR) through the Medical Research Coordinating Committee (MRCC), Ref: NIMR/HQ/R.8a/Vol. IX/3495. Before the commencement of the study, meetings were held with volunteers who participated in mosquito collection, where the purpose and procedures of the study were explained. The consent forms were then signed by the volunteers who agreed to participate in the study.

Consent for publication

This manuscript has been approved for publication by the National Institute of Medical Research, Tanzania (NIMR/HQ/P.12 VOL XXXII/64).

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Wild Populations of Malaria Vectors Can Mate Both Inside and Outside Human Dwellings

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