BACKGROUND: There is a paucity of data regarding resting habits and comparison of the physiological states of Anopheles mosquito populations from indoor and outdoor collection venues in Ethiopia. Therefore, this study aims to investigate resting behavior and age structure of the malaria vectors in Wonji Sugar Estate.
METHOD: Longitudinal entomological study was conducted at Wonji Sugar Estate from July 2018 to June 2020. Pyrethrum spray catches (PSC), Pit Shelter (PIT), and Oral Aspirators were used to collect adult Anopheles species from various resting sites. Mosquitoes were identified to the species using morphological keys. Finally, the resting behavior of Anopheles mosquitoes were determined using standard protocol.
RESULT: A total of 1406 mosquitoes were collected from indoor and outdoor resting venues, with the outdoor resting population being significantly higher (F = 8.296, P = .04). The number of half-gravid/gravid Anopheles species collected from outdoors was higher than that from indoor resting venues (F = 35.612, P = .00). Eight two percent of the indoor sampled Anopheles species left the indoor venue before completing their gonotrophic cycle. Anopheles pharoensis and An. coustani cx. exhibited exophily rate of 83.4% and 100%, respectively. The overall parity rate of Anopheles gambiae s.l. was 45.7%. The highest parity rate, daily survival rate, and longevity of Anopheles gambiae s.l. was 66.7%, 0.874, and 7.399 days, respectively. Anopheles gambiae s.l. reached the infective stages for Plasmodium falciparum and P. vivax in February 2019.
CONCLUSION: Anopheles mosquitoes, including the major malaria vector Anopheles gambiae s.l., showed predominantly exophilic habits, underscoring the need for outdoor vector control strategies in addition to indoor-based interventions. The increase in the nulliparous population indicates a higher risk of vector-human contact. Meanwhile, the extended lifespan of Anopheles gambiae s.l. observed during the dry season could increase the risk of infectious bites.
Introduction
Anopheles gambiae sensu lato, the primary malaria vector in Ethiopia, is known for its plastic behaviors in host choice, feeding, and resting site preferences.1-4 On the other hand, Anopheles pharoensis and Anopheles funestus, secondary malaria vectors, are known for their outdoor and indoor biting behaviors, respectively. This difference in behavior possibly makes them more efficient in facilitating residual malaria transmission, which poses a challenge to current intervention tools.5-7 The core vector control measures in use in Ethiopia are long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). These strategies reduce the risk of malaria infection by targeting indoor biting and resting mosquitoes.8-10 However, studies from the country indicate that malaria vectors have undergone behavioral modifications, potentially in response to the scale-up of indoor-based malaria vector control tools.9,11,12 Moreover, these tools do not target outdoor biting and resting mosquitoes.12,13 For instance, Anopheles species like Anopheles pharoensis, Anopheles nili, and Anopheles coustani cx., which are primarily exophagic and exophilic, play a role in malaria transmissions in the country as secondary vectors. Current control tools are ineffective against such species.2,7,14,15
Furthermore, studies from southwest and south-central Ethiopia have shown that outdoor mosquito human-biting activities surpass indoor ones during the early and late parts of the night, especially at dusk and dawn when human activity is higher2,3 in the interventional periods. It is believed the anopheline’s behavioral heterogenicity, in the presence of vector control tools presumably sustains residual malaria transmission, thereby failing to protect against outdoor transmission.3,15-18 Moreover, there is concern that achieving the country’s malaria elimination strategic plan by 2030 will face challenges.19
Similarly, in sub-Saharan Africa and other WHO malaria-endemic regions, modifications in the behavior of malaria vectors are challenging the efficacy of indoor-based vector control strategies.11,12,20 These behavioral modifications include; changes in biting time and location,2,3,15,16,21 shifts from anthropophilic to zoophilic host choice or becoming opportunistic, and changes in resting behavior from endophilic to exophilic.13,17 For example, shifts in feeding behaviors from endophagic to exophagic or opportunistic have been reported in connection with the long-term use of insecticide-based indoor interventions.3,16 Studies on members of the notorious malaria vectors Anopheles gambiae s.l. and An. funestus across their geographic distribution in sub-Saharan Africa including Ethiopia, have reported that they tend to shift from endophagic and endophilic behaviors to exophagic and exophilic ones to the selected IRS and ITNs selective pressures.6,21,22 For this reason, their longevity is not affected, and thus, they continue to contribute to residual malaria transmission, although they were previously reported and well-known for being endophilic, endophagic, anthropophilic, and late-night biters.6,23
In communities where both LLINs and IRS are in use, it is important to assess the resting behavior and age structure of mosquitoes. This is particularly relevant in cane farms, where factors such as overnight outdoor human activities and year-round potential breeding habitats influence vector behaviors. Unfortunately, there have been few studies on the outdoor resting behavior of Anopheles species at natural and pit shelters, both within the study site and nationwide.13 Debebe et al13 conducted exceptional studies investigating the degree of exophilic preferences among Anopheles species’ from outdoor collections. Meanwhile, 2 other studies conducted in western and central parts of the country have addressed the resting behavior focused on blood digestion stages of the local Anopheles species, sampled from indoor resting venues.6,7 It is unfortunate that no study has comprehensively examined the physiological states of mosquitoes in both indoor and outdoor venues in Ethiopia. Thus, this study aims to fill existing knowledge gaps and provide evidence for more effective vector control interventions.
Methods
Description of the study area
This study was carried out at the Wonji Sugar Estate (Figure 1). The estate encompasses 9 villages embedded within the cane farms. The estate is home to 48 000 residents. Approximately 38% of the residents are office workers who live in facilitated houses with metal roofing and cement walls in 2 central villages. The majority of the residents are grassroots workers, including seasonal and casual laborers, living in houses with metal roofs and mud-plastered woody walls in 9 villages. The employees come from various geographic, educational, and economic backgrounds.24,25 According to the researcher’s observations and available data, some camp dwellers own 1 to 3 domestic animals such as cattle, goats, sheep, hens, dogs, and cats. These animals share the burden of Anopheles species and culicine blood meals with the community. The residents have access to 1 primary hospital, 1 polyclinic, and 2 post-basic health facilities. The estate’s malaria control unit report indicates that the estate has achieved 100% distribution of LLINs and 90% IRS coverage, which are 20% and 10% higher than the district’s coverages respectively. However, residual malaria transmission continues to challenge the community’s health and well-being. Recent studies by Lelisa et al24 found an 11.74% malaria-positivity rate in the community,24 surpassing the national standard for districts designated as having low malaria transmission. The Wonji Sugar Estate is among the villages targeted in the country’s malaria elimination plan. Unfortunately, persistent potential mosquito breeding habitats created by intensive cane farming for the sugar industry have exacerbated the situation by favoring the availability and survival of malaria vectors in the community.24 The area experiences its longest dry season from October to February and May, with short and long rainy periods occurring in March, April, and June through September.
Mosquito collection
A longitudinal entomological study was conducted to determine the resting site preferences and the age structure of the major malaria vector, Anopheles gambiae s.l., from July 2018 to June 2020.
Adult Anopheles mosquitoes were sampled from 2 randomly selected villages (Wonji Shoa and Bikiltu) from among the 9 villages that have similar exposure to cane fields. Thirty households were purposely selected for entomological sampling in both villages, with 15 households from each, considering inter-household breeding site distance, and mosquitoes flight ranges.26 Adult Anopheles mosquitoes were collected from indoor resting sites using pyrethrum spray catches (PSC) for indoor resting mosquitoes. Handheld mouth aspirator used for outdoors naturally available shady resting sites (N) and artificially prepared pit shelters (PIT). Every month, the same households were inspected to compare the indoor and outdoor resting habits of Anopheles mosquitoes. The households selected for indoor resting collections were also used for outdoor inspections. The indoor and outdoor resting sites were located within an average radius of 15 to 25 m from each other.27 Assessments were conducted simultaneously for both indoor and outdoor resting sites, starting at 6:00 AM and ending at 07:30 AM. Collectors spent an average of 15 to 20 minutes in each household both for indoor and outdoor collections.26 Householders were asked for permission to sample resting mosquitoes in their homes and compounds 1 day before each collection.
Assessment of indoor and outdoor resting Anopheles mosquito
Twenty households were sampled for indoor and outdoor resting mosquitoes from both villages, 10 households from each. Before implementing the PSC, household items such as food, water, domestic animals, and other items were brought out. Any openings that could allow mosquitoes to escape were checked and sealed. The floor was completely covered with a white cotton sheet. The house was then sprayed with Mobil flit (KillitMT insecticide aerosol), which contains pyrethroids, by a person wearing protective gear, and subsequently locked for 10 minutes. After ventilating the houses for about 5 minutes, the sheet was gently removed from the room, and knocked-down mosquitoes were collected with forceps.26
For the collections using hand-held mouth aspirators, all naturally available outdoor shady resting sites were assessed. The identified shady sites included leftover household materials stored in the compound, tree bark, tree holes, animal shelters, and sides of outdoor walls that could serve as resting spaces for the anophelines ( Supplemental Figure 1a-d). In some households, multiple resting sites were detected.
For the artificial pit shelter collections, 20 pits were prepared in potentially shady areas within the compounds encompassing both indoor PSC and outdoor natural resting sites. Each pit was 1.5 m deep with an opening measuring 1.2 m by 1.2 m sizes. Additionally, each pit shelter contained 4 digs/pockets, each 30 cm deep located on each side.26,28 To prevent children and animals from falling into the pits, they were enclosed with visible barriers that were not entirely sealed; openings were left for mosquito entry. LLINs were used to prevent mosquitoes from escaping during collection. By the end of the study, all pit shelters had been closed ( Supplemental Figure 1e-h).
Assessment of indoor and outdoor biting Anopheles mosquito
This tool was used to survey unfed mosquitoes, to determine their age following dissection. Accordingly, the Center for Disease Control and Prevention light trap (CDC LT) (Model 512; John W. Hock Co., Gainesville, FL, USA) was set up both indoors and outdoors in 10 households, with 5 households in each village. The CDC LT operated from 18:00 to 6:00 and was used twice a month at fortnight intervals.27
Anopheles mosquito processing
Species identification.
Sampled adult Anopheles mosquitoes were morphologically identified using a standard key29; no molecular identification was performed for species complexes. Specimens were labeled and preserved with silica gel in 2.5 ml Eppendorf tubes each labeled with information including species type, date of collection, place of collection, method of collection, and abdominal status. The specimens were stored in a freezer at −40°C at the Aklilu Lemma Institute of Pathobiology Insectary laboratory for subsequent activities.
Abdominal status detection.
The abdominal status of the Anopheles mosquitoes was assessed according to the methods previously described26 and categorized into 4 physiological states: unfed (UF), freshly fed (F), half-gravid (HG), and gravid (G). The parameters were evaluated for both indoor and outdoor resting sites, to determine the proportion of surveyed Anopheles populations that had fed the previous night. This also allowed for the study of their post-feeding resting behaviors until completing their first gonotrophic cycle.6,7,13,26
Determination of degree of exophily or endophily.
The proportion of fed (F) to half-gravid/gravid (HGG) Anopheles species sampled from the resting venues was used in determining resting habits.6,7,13 The rationale behind the F: HGG ratio is that Anopheles species, after blood meals, will stay where they were surveyed until oviposition and the next blood meal, with the ratio becoming 1:1.30
Therefore, the degree of exophily or endophily was calculated from this ratio as6:
Age determination of Anopheles gambiae s.l.
Adult female An. gambiae s.l. samples, trapped from biting activities by CDC LT and from resting sites by PSC, PIT, and hand-held mouth aspirators were sorted according to abdominal blood digestion stages into unfed, fed, half-gravid, and gravid. The unfed samples were dissected under a dissecting microscope following the Detinova method for age determination.31 The dissected ovaries were left to dry and were examined under a compound microscope (10x and 40x magnification). The observed ovary was determined to be either nulliparous or parous based on tracheolar skeins. An ovary was declared parous when the observed tracheolar skeins had uncoiled endings ( Supplemental Figure 2a). If the mosquito’s ovarian tracheoles had tightly coiled endings, the ovary was determined to be nulliparous ( Supplemental Figure 2b).
Following ovarial dissection, the vector’s age and the extrinsic incubation periods of the parasites within the vector were determined. Accordingly, parous rates (PR) (
), daily survival rate (P) 
, and longevity (L) 
were determined.31 Where; NE = the total number examined, NP = the number of parous detected, gc = the Estimated gonotrophic cycle, ln = the natural logarithm of the constant 2.7183.
The probability of daily surviving sporogony (S) in Anopheles gambiae s.l. (S = Pn), for P. falciparum and P. vivax was determined. Where P = probability of Anopheles gambiae s.l. population surviving a day n = the duration of sporogony (extrinsic incubation periods/EIP) from time of ingestion to infective bite which was determined from 
following Moshkovsky protocol in WHO 1975.26 Where b = 111 and 105 temperature degrees–days for P. falciparum and, P. vivax respectively, c = the difference between mean temperature per time and the threshold temperature for extrinsic development of Plasmodium parasites, which was given as 16°C for P. falciparum and 14.5°C for P. vivax. Accordingly, the duration of sporogony is calculated as; n = 
for Pf., and 
for Pv. Where T°C is the environmental temperature obtained from the metrological agency of the estate.
Meteorological Data
Meteorological data was obtained from the Wonji Sugar Estate meteorological station.
Data analysis
Data were analyzed using SPSS statistical software package version 26 (IBM, Corp., Chicago, IL). A probability (P-value) at <.05 was considered statistically significant. Before analysis, data cleaning and log (x + 1) transformations were performed to normalize the distribution of count data. Multiple comparisons of the mean monthly densities of Anopheles species’ per trap/house/night for indoor and outdoor resting were analyzed using 1-way ANOVA, and significant means were separated using the Tukey post hoc test. Two-way ANOVA was used to detect the influence of seasons on the resting behavior. The F-test was used to examine variation among Anopheles collection tools, while the degree of exophily was determined following Ameneshewa and Service.7 The parity rate, probability of daily survival, longevity, and probability of sporogony surviving a day were determined following standard protocol.31
Results
Indoor and outdoor resting density
From PSC, PIT, and oral aspirator tools used for collecting resting mosquitoes indoors and outdoors, 1406 individual Anopheles mosquitoes representing 3 species: Anopheles gambiae s.l., Anopheles pharoensis, and Anopheles coustani cx. were collected. Statistical analysis showed a significant variation in mean density of Anopheles mosquitoes indoors and outdoors (F = 8.296, P = .04) (Table 1). The mean of outdoor collection (mean = 0.199 ± 0.009) was higher than that of indoor resting (mean = 0.154 ± 0.013), despite the lack of a significant difference in the mean density of Anopheles gambiae s.l. between indoor and outdoor resting venues (F = 0.190, P = .890). In contrast, the mean densities of Anopheles pharoensis and Anopheles coustani cx. collected from outdoors was greater than indoor resting venue (P = .000). However, the indoor and outdoor resting densities of Anopheles pharoensis and Anopheles gambiae s.l. exhibited inconsistency throughout the study period ( Supplemental Information III).
Table 1.
Anopheles mosquito species mean density variation from indoor and outdoor resting sites.
A significantly higher number of Anopheles mosquito species were captured from outdoor natural resting sites (mean 0.245, n = 644) compared to those caught in pit shelters (mean = 0.152, n = 340). This difference was statistically significant (F = 55.193, P = .000) (Table 2).
Table 2.
Anopheles species mean density variation among artificially prepared pit shelters and naturally available outdoor resting sites.
Figure 2 presents count data of Anopheles species from their resting venues.
Seasonal variation of indoor and outdoor resting behavior
It is interesting to note that the behavior of local Anopheles species defined in terms of indoor and outdoor resting preferences remained consistent across seasons, as indicated by a 2-way analysis of variance comparing the wet and the dry seasons (df = 1, F = 3171, P = .075). In both seasons, a greater number of mosquitoes were identified to rest outdoors than indoors although more Anopheles mosquitoes were captured during the wet season than the dry season (df = 1, F = 236.962, P = 0.00).
Anopheles mosquitoes’ physiological state
According to the analysis of the abdominal blood digestion status of Anopheles mosquitoes collected from indoor and outdoor resting venues, a significant number of the population was fed, followed by half-gravid/gravid individuals, whereas unfed were the least common (Figure 3). The half-gravid/gravid populations of Anopheles species collected from the outdoors were significantly higher than the indoor resting venue (F = 35.612, P = .00). More importantly, the unfed Anopheles mosquito species collected indoors and outdoors didn’t show significant variation (F = 0.293, P = .588). However, significantly higher fed Anopheles species were collected from indoor than outdoor venues (F = 49.944, P = .00).
Figure 3.
Physiological states of Anopheles species by resting venues, Wonji Sugar Estate, Oromia, Ethiopia.
Abbreviations: F, fed; HGG, half-gravid/gravid; UF, unfed.
The half-gravid/gravid population of Anopheles gambiae s.l. by resting venues indicates significant variation (P < .001), with a higher population being collected from outdoor resting venue. No gravid Anopheles coustani cx. was collected from indoor resting sites.
Table 3 shows that the probability of collecting a fed population was 5.7 for every half-gravid/gravid Anopheles gambiae s.l. resting indoors. In contrast, the outdoor ratio of fed to half-gravid/gravid population indicates approximately equal probability.
Table 3.
Resting habits of Anopheles species from the analysis of abdominal blood digestion status.
Exophily and endophily behaviors of Anopheles mosquito
Figure 4 depicts the exophilic and endophilic behavior of Anopheles species assessed from indoor and outdoor resting venues. Accordingly, 82% of the Anopheles species populations that rest indoors depart before completing their gonotrophic cycle. On the other hand, 70% of Anopheles species collected from outdoor resting venues remain outdoors until completing their gonotrophic cycle. Moreover, the overall data analysis from indoor and outdoor resting venues showed that 49.7% of Anopheles species stayed at the venues where they were surveyed until they moved to the oviposition site.
Figure 4.
Degree of exophily and endophilic determined from indoor and outdoor resting Anopheles species, Wonji Sugar Estate, Oromia, Ethiopia.
According to the blood digestion status of Anopheles gambiae s.l. population caught from outdoor resting venue, 62.5% preferred to remain outdoors. Anopheles coustani cx. had less probability of staying indoors to complete the gonotrophic cycle in the indoor venue as the relative proportion of fed to half-gravid/gravid indicates. Similarly, Anopheles pharoensis exhibits a greater degree of exophilic behavior, with 90.9% of the blood-engorged population having a probability of remaining outdoors until oviposition and the subsequent blood meal (Table 3).
Parity rate determination and survival rate of Anopheles gambiae s.l.
Of 591 unfed Anopheles gambiae s.l. dissected, 270 individuals were parous (Table 4) with an overall parity rate of 45.7%. Approximately, half of the population had practiced hematophagy at least once before they were trapped. In April 2019 and February 2020, the highest parity rate (66.7%), probability of daily survival (0.874), and longevity (7.399 days) of Anopheles gambiae s.l. was noticed. In contrast, the lowest parity rate, daily survival rate, and longevity were observed in September 2019. The mean minimum and maximum probability of daily survival rate of Anopheles gambiae s.l. population ranged from 0.613 to 0.874, respectively (Table 4).
Table 4.
Parity rate, probability of daily survival rate, and longevity of Anopheles gambiae s.l.
Anopheles gambiae s.l. age structure by seasons
The parity rate of Anopheles gambiae s.l. was higher in dry seasons than in rainy seasons (F = 35.58, df = 1, P < .05). The 2018 rainy season showed higher parity rates, daily survival, and longevity than the 2019 rainy season (Table 5).
Table 5.
Parity rate, probability of daily survival rate, and longevity of Anopheles gambiae s.l. across seasons.
Determination of Plasmodium species extrinsic incubation periods in Anopheles gambiae s.l.
The highest probability of Anopheles gambiae s.l. daily surviving the sporogony of Plasmodium falciparum and P. vivax was 0.385 and 0.438, respectively in February 2019. In contrast, the lowest probability of daily surviving sporogony for P. falciparum and P. vivax was 0.008 and 0.017, respectively in September 2019. The maximum number of days required for the development of malaria parasites in the vector from gametocytes to sporozoites, that is, from the day ingestion to infectious bite, was 11.65 and 9.52 in December 2019 for P. falciparum and P. vivax, respectively. During the 24-month study period, the age of Anopheles gambiae s.l. capable of completing the extrinsic incubation periods of P. falciparum and P. vivax was determined only in February and April 2019, February and March 2020. The results suggest that the mosquitoes were mature enough to complete the extrinsic incubation periods of the 2 parasites in these months (Table 6). During the study periods, the minimum temperature observed was 25.53°C in August 2018, and the maximum was 31.7°C in February 2019, with an annual mean temperature of 28.5°C.
Table 6.
Probability of surviving sporogony of Plasmodium species in An. gambiae s.l. by month.
Discussion
The indoor and outdoor resting behavior of Anopheles species and their physiological states/gonotrophic cycles were determined. Based on the findings potential, challenges to the indoor-based vector control tools in use in the community were discussed in this section. The results indicated that Anopheles gambiae s.l., the primary malaria vector in Ethiopia, prefer both indoor and outdoor resting venues, which is consistent with previous findings.23,32 However, analysis of the species’ physiological state revealed a notable inclination toward exophilic behavior. In this study, 14.93% of the Anopheles mosquitoes completed their gonotrophic cycle indoors, while 85.07% left houses and complete their gonotrophic cycle outdoors. This might be a behavioral change due to the effect of indoor-based interventions. Furthermore, to confirm this movement, a comparison of the proportion of fed Anopheles gambiae s.l. to the half-gravid/gravid population collected by PIT and N outdoors was evaluated which showed a ratio of 1.6:1. Krafsur evaluated such movement in the course of gonotrophic cycle accomplishment in Gambella region in southwest Ethiopia.6 There could be several reasons as to why Anopheles gambiae s.l. exhibited exophilic behavior in the community: (1) Exerted pressure of indoor-based vector control tools IRS and ITNs excito-repellence effect, (2) Indoor disturbance, (3) Due to the presence of shady spaces with ambient humidity outdoors. The suggested possible scenarios observed in the study community were also supported by different scholars’ finding.6,7,13,33
In general, 82% of Anopheles species left the indoor resting venues within 48 to 72 hours before completing their gonotrophic cycle; which is in agreement with Krafsur in Southwest Ethiopia that showed 24% to 60% of Anopheles species rest indoors, leaving the place before completing their gonotrophic cycle.6 The results of this study is also in agreement with a study by Debebe et al, which reported that the majority of Anopheles species in clay pots were half-gravid or gravid in their physiological state.13
The observed behavioral modification could hinder the country’s progress toward elimination in low transmission districts where both IRS and ITNs are implemented.23 A study conducted in South-eastern Tanzania revealed intriguing findings similar to ours, in which Anopheles mosquito learned to avoid vector control interventions by resting on various surfaces at home, such as utensils, flowers, roofs, and clothes, which had not been treated with IRS for several reasons.34
Characterization of Anopheles gambiae s.l.’s indoor and outdoor resting preferences and physiological states during the gonotrophic cycle in this study presented interesting insights into malaria vector ecology, that call on/triggers/ for additional bionomics studies using mark release recapture as well as molecular assays. Moreover, understanding the physiological state of mosquitoes during the gonotrophic cycle is crucial for designing effective interventions to disrupt their reproductive success.
Anopheles pharoensis and Anopheles coustani cx., underscore exophilic behavior, that agrees with studies conducted in different regions of Ethiopia.32,35 The occurrence of Anopheles pharoensis alongside local populations’ outdoor activities in the estate may elevate the risk of vector-human contact, potentially leading to infectious mosquito bites. This species has established a secondary role in malaria transmission. Thus, it is crucial to closely monitor their contribution to residual malaria transmission within the community, as indoor-based vector control tools may not fully target them.20,34,36 Regarding Anopheles coustani cx., only an insignificant number of them were collected indoors by PSC. However, in the outdoor PIT and N collections, the proportion of the fed population to the halfgravid/gravid showed a strong degree of exophilic behavior, which agrees with Lelisa et al’s5 report from southwest Ethiopia.5
In addition to studying mosquito resting behavior, this study also investigated the age of Anopheles gambiae s.l., a major malaria vector in the country. The age determination of mosquitoes is crucial for understanding malaria transmission dynamics and controlling its spread.31 This is particularly relevant on cane farms that rely on conventional irrigation for sugar cane production, as it directly impacts the lifespan of the vector. This study finds a higher number of newly emerging populations of Anopheles gambiae s.l. depicted from ovaries dissection being nulliparous (n = 321) compared to the aged parous population detected. Similarly, studies conducted in irrigation-based cane farms in the country by different scholars confirmed a proliferation of the newly emerging mosquito population and enhanced ages of Anopheles gambiae s.l.37,38 This could be seen as the pros and cons of cane farming were the shorter the lifespan detected could contribute to a low infectivity rate, whereas the newly added population increases the risk of human vector contact rate.
The overall life expectancy of Anopheles gambiae s.l. determined in this study was 4.66 days; while 2.046 days, was the mean minimum and 7.399 days was the mean maximum life expectancy. It implies, that there is less probability of an infective bite in the community, but the chance of an infective bite was not null. The result was comparable with the finding of Taye et al’s who reported a 6.5 life expectancy for Anopheles gambiae s.l. in Lare district, Gambella, Ethiopia.39 The observed result might underestimate the aged population which is the limitation of this study. Alternative age estimation methods have to be used for accurate estimation like adult survivalship experiment and mark release recapture method.7,37 On the other hand, the elderly population might be influenced by the prolonged use of pesticides and herbicides and exposure to indoor-based vector controls in the community.24 This result disagrees with the study findings of Gari et al, which determined the average age of Anopheles arabiensis as 14 days, and Hawaria et al which found older Anopheles gambiae s.l. sufficient to enhance the development of extrinsic incubation periods and infectious bites.37,40
Seasonally, the oldest age of Anopheles gambiae s.l. was determined in the dry rather than the rainy season. Similarly, a study by Kibret et al41 determined a more aged population during the dry season.41 This was probably the contribution of the presence of potential breeding habitats due to the cane farm. The researcher’s observation indicates that there was also mismanagement of canals in the cane farm, which supported mosquito density in dry seasons and longer survival for Anopheles gambiae s.l. The study by Tufa also confirms the lack of canal water management in the estate.25 The scenario sustains annual malaria residual transmission in the community,24 contradicting the seasonal malaria transmission patterns in the country.1
Moreover, the current study indicated the higher probability of daily surviving sporogony in the early months of dry seasons, where the maximum life expectancy of Anopheles gambiae s.l. was determined, which was consistent with other studies.42,43 A possible reason could be that the extrinsic incubation periods are highly influenced by temperature, as observed in February 2019. As the temperature increases, the extrinsic incubation periods for parasite development, spanning from the initial gametocyte ingestion to the onset of infectious bites, become shorter. This scenario indicates a risk of malaria transmission in the study community, and it also overlaps with the highest malaria transmission periods in the country.1
Conclusions
In light of the findings on the physiological states to express resting behavioral patterns of Anopheles gambiae s.l., the species demonstrated a high degree of exophilic behavior. While the density-based indoor and outdoor sampled data showed flexibility in locating resting sites, such data might not infer how long the species stay at the venues where surveillance has been conducted. Therefore, it is imperative to complement density-based assessments of resting habits determination with additional entomological parameters to evaluate the effectiveness of vector control strategies. Anopheles coustani cx. and Anopheles pharoensis have shown strict exophilic behavior both in dry and wet seasons. The high population of Anopheles gambiae s.l. determined as a new requirement from the age structure implies the contribution of potential breeding habitats due to cane farms as well as the impact of indoor-based vector control tools in use in the community. Anopheles gambiae s.l. showed an extended life span in dry seasons than in rainy seasons, resulting in infective bites and retain residual malaria transmission in the community.
Acknowledgements
We are grateful to Addis Ababa University and Dilla University for funding this study. We would like to thank Oromia Health Bureau, East Shoa Zone, and Nanawa Adama District’s Health Office for their support and for facilitating the study. We are grateful to Wonji Sugar Estate for their enormous support, which includes access to the laboratory and mereological data, housing, and facilitating the study in the estate. We are indebted to the households where mosquitoes were collected. We sincerely thank the entomological data collector team. The critical comments and suggestions provided by reviewers have made a great contribution to this work to be easily understandable for readers therefore, they deserve our great appreciation.
© The Author(s) 2024
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Author Contributions
KL conceived the study design, undertook the field study, performed data analysis, interpreted data, and drafted the manuscript. SD is involved in the study design, revision of the manuscript, and facilitation of administrative issues. SD, YW, and LG take part in a critical review of the manuscript for intellectual content. All authors read and approved the final manuscript.
Ethical Approval and Consent to Participate
Ethical clearances were obtained from Addis Ababa University, Aklilu Lemma Institute of Pathobiology, Institutional Review Board (IRB) (Reference number: ALIPB/IRB/012/2017/2018), and from Oromia Regional Health Bureau Ethical Clearance Committee (Reference number: BEFO/DBTHL/1-8/422). A letter of permission was also sought from the East Shoa zone and Nanawa Adama district’s health office for the commencement of the study. Wonji Sugar Estate provided us permission and led us to the community level where we sought their consent. Every month data collection permission was sought from each house owner before the commencement of entomological sampling and during PSC they consented to stay outside for about 20 minutes post-spray.
Availability of Data and Material
The data supporting the results reported in this article are well addressed within this article and in supplemental information annexed.
Supplemental Material
Supplemental material for this article is available online.
