Malaria Vectors Tend to Avoid the Indoor-Based Vector Control Tools in Wonji Sugar Estate,Oromia,Ethiopia

Abstract

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.

Methods:

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.

Results:

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.

Conlusion:

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.

Keywords

Malaria vectors exophily endophily longevity oromia Ethiopia

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 higher^2,3 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.

Figure 1.

Map of the study area.

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:

1 - (\frac{1}{F : H G G}) 100 .

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) ( $P R = \frac{N P}{N E}$ ), daily survival rate (P) $(P = \sqrt[g c]{P R})$ , and longevity (L) $(L = \frac{1}{- \ln p}$ ) 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. ( $S = P^{n}$ ), 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 ( $n = \frac{b}{c}$ ) 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 = $\frac{111}{T ° C - 16 ° C}$ for Pf., and $\frac{105}{T ° C - 14.5 ° C}$ 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.

Anopheles species	n	Indoor/PSC (M ± SE)	Outdoor/PIT & N (M ± SE)	F-test	P-value
1. Anopheles gambiae s.l.	1093	0.140 ± 0.012	0.146 ± 0.008	0.190	.890
2. Anopheles pharoensis	244	0.019 ± 0.004	0.051 ± 0.005	85.086	.000
3. Anopheles coustani cx.	69	0.001 ± 0.001	0.015 ± 0.003	51.929	.000
Total	1406	0.154 ± 0.013	0.199 ± 0.009	8.296	.04

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.

Anopheles species	n	Outdoor/PIT (M ± SE)	Outdoor/N (M ± SE)	F-test	P-value
1. Anopheles gambiae s.l.	706	0.112 ± 0.010	0.180 ± 0.013	65.778	.000
2. Anopheles pharoensis	211	0.044 ± 0.006	0.058 ± 0.007	8.265	.004
3. Anopheles coustani cx.	67	0.006 ± 0.002	0.025 ± 0.005	52.738	.000
Total	984	0.152 ± 0.011	0.245 ± 0.014	55.193	.000

Abbreviation: n, number collected.

Figure 2 presents count data of Anopheles species from their resting venues.

Figure 2.

Number of Anopheles species trapped from indoor and outdoor resting venues, Wonji Sugar Estate, Oromia Ethiopia.

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.

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.

Species	Tools	Venue	Ratio of F:HGG	DE = $1 - (\frac{1}{F : H G G}) 100$ (%)
Anopheles. gambiae s.l.	PSC	In	5.7:1	82.5
Anopheles. gambiae s.l.	PIT&N	Out	1.6:1	62.5
Anopheles. pharoensis	PSC	In	6:1	83.4
Anopheles. pharoensis	PIT&N	Out	1.1:1	90.9
Anopheles. coustani cx.	PSC	In	NA	100
Anopheles. coustani cx.	PIT&N	Out	0.73:1	100
Total	PSC	In	5.72:1	82
Total	PIT&N	Out	1.4:1	70
Ground total			1.99:1	49.7

Abbreviations: DE, degree of exophily; NA, not applicable.

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.

Month	#Disected	#nullparous	#Parous	Parity rate (%)	Daily survival	LE (in days)
2018 July	32	19	9	28.1	0.655	2.365
2018 August	23	9	11	47.8	0.782	4.067
2018 September	58	30	26	44.8	0.765	3.739
2018 October	28	14	10	35.7	0.709	2.914
2018 November	21	9	7	33.3	0.693	2.731
2018 December	8	5	3	37.5	0.721	3.059
2019 January	10	5	5	50	0.794	4.328
2019 February	3	0	2	66.7	0.874	7.399
2019 March	9	4	5	55.6	0.822	5.104
2019 April	6	2	4	66.7	0.874	7.399
2019 May	11	5	6	54.5	0.817	4.949
2019 June	81	49	30	37	0.718	3.020
2019 July	46	27	19	41.3	0.745	3.393
2019 August	31	20	11	35.5	0.708	2.895
2019 September	26	16	6	23.1	0.613	2.046
2019 October	13	7	6	46.2	0.772	3.880
2019 November	11	4	7	63.6	0.860	6.637
2019 December	6	3	3	50	0.794	4.328
2020 January	20	7	13	65	0.866	6.964
2020 February	9	3	6	66.7	0.8736	7.399
2020 March	17	6	11	64.7	0.865	6.89
2020 April	42	14	24	57.1	0.830	5.361
2020 May	34	11	20	58.8	0.838	5.654
2020 June	46	16	26	56.5	0.827	5.258
Total	591	285	270	45.7	0.770	3.829

Abbreviation: LE, life expectancy/longevity.

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.

Seasons in year	Anopheles gambiae s.l.					Mean	F-test	SE	P-value
Seasons in year	ND	Parous	PR (in %)	P	LE (in days)	Mean	F-test	SE	P-value
Rainy 2018	113	46	40.7	.741	3.338
Dry 2018	57	20	35.1	.705	2.864
Rainy 2019	201	76	37.8	.723	3.085
Dry 2019	52	28	53.8	.814	4.846
Rainy 2020	122	70	57.4	.831	5.400
Dry 2020	46	30	65.2	.867	7.018
Rainy	436	192	44	.761	3.658	0.016	35.58	0.01	0
Dry	155	78	50.3	.795	4.369	0.042	35.58	0.01	0
Total	591	270	45.7	.770	3.829

Abbreviations: LE, life expectancy; ND, total number dissected; P, probability of daily survival rates; PR, parous rate.

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.

Month	P	LE	T°C	n of Pf.	n of Pv.	S of Pf.	S of Pv.
July 2018	.655	2.365	26.758	10.318	8.566	0.013	0.027
August 2018	.782	4.067	25.818	11.306	9.277	0.062	0.102
September 2018	.765	3.739	28.561	8.837	7.467	0.094	0.136
October 2018	.709	2.914	28.968	8.560	7.258	0.053	0.083
November 2018	.693	2.731	27.945	9.292	7.809	0.033	0.057
December 2018	.721	3.059	27.500	9.652	8.077	0.043	0.071
January 2019	.794	4.328	27.950	9.289	7.807	0.117	0.165
February 2019	.874	7.399	31.710	7.066	6.101	0.385	0.438
March 2019	.822	5.104	31.370	7.222	6.224	0.243	0.295
April 2019	.874	7.399	30.860	7.470	6.418	0.364	0.420
May 2019	.817	4.949	31.010	7.395	6.360	0.224	0.277
June 2019	.718	3.020	29.300	8.346	7.095	0.063	0.095
July 2019	.745	3.393	26.560	10.511	8.706	0.045	0.077
August 2019	.708	2.895	26.280	10.798	8.913	0.024	0.046
September 2019	.613	2.046	27.100	10.000	8.333	0.008	0.017
October 2019	.773	3.880	26.700	10.374	8.607	0.069	0.109
November 2019	.860	6.637	26.900	10.183	8.468	0.216	0.279
December 2019	.794	4.328	25.530	11.647	9.519	0.068	0.111
January 2020	.866	6.964	27.100	10.000	8.300	0.238	0.304
February 2020	.874	7.399	29.300	8.346	7.095	0.324	0.383
March 2020	.865	6.892	31.100	7.351	6.325	0.344	0.399
April 2020	.830	5.361	30.000	7.929	6.774	0.228	0.283
May 2020	.838	5.654	30.000	7.929	6.774	0.246	0.302
June 2020	.827	5.258	30.230	7.800	6.675	0.227	0.281
Mean	.78	4.66	28.52	9.07	7.62	0.16	0.20

Abbreviations: LE, life expectancy; n, extrinsic incubation periods; P, probability of daily survival rate; Pf., Plasmodium falciparum; Pv., Plasmodium vivax; S, probability of surviving sporogony; T°C, atmospheric temperature.

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.

Supplemental Material

sj-docx-1-ehi-10.1177_11786302241267186 – Supplemental material for Malaria Vectors Tend to Avoid the Indoor-Based Vector Control Tools in Wonji Sugar Estate, Oromia, Ethiopia

Supplemental material, sj-docx-1-ehi-10.1177_11786302241267186 for Malaria Vectors Tend to Avoid the Indoor-Based Vector Control Tools in Wonji Sugar Estate, Oromia, Ethiopia by Kidane Lelisa, Lemu Golassa, Yitbarek Woldehawariat and Sisay Dugassa in Environmental Health Insights

Footnotes

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.

Funding:

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Addis Ababa and Dilla University have financially supported the study. However, the organizations have no role in the design, data collection, analysis, interpretation of the data, and decision to publish.

Declaration of conflicting interests:

The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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.

ORCID iD

Kidane Lelisa

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.

References

Federal Ministry of Health. Ethiopia Malaria Elimination Strategic Plan 2021-2025. Ministry of Health; 2020.

Taye

Lelisa

Emana

Asale

Yewhalaw

Seasonal dynamics, longevity, and biting activity of anopheline mosquitoes in southwestern Ethiopia. J Insect Sci. 2016;16:6.

Degefa

Githeko

Lee

Yan

Yewhalaw

Patterns of human exposure to early evening and outdoor biting mosquitoes and residual malaria transmission in Ethiopia. Acta Trop. 2021;216:105837.

Kibret

Wilson

GG.

Increased outdoor biting tendency of Anopheles arabiensis and its challenge for malaria control in Central Ethiopia. Public Health. 2016;141:143-145.

Lelisa

Asale

Taye

Emana

Yewhalaw

Anopheline mosquitoes behaviour and entomological monitoring in southwestern Ethiopia. J Vector Borne Dis. 2017;54:240-248.

Krafsur

The bionomics and relative prevalence of Anopheles species with respect to the transmission of Plasmodium to man in western Ethiopia. J MedEntomol. 1977;14:180-194.

Ameneshewa

Service

MW.

Resting habits of Anopheles arabiensis in the awash river valley of Ethiopia. Ann Trop Med Parasitol. 1996;90:515-521.

Taffese

Hemming-Schroeder

Koepfli

, et al. Malaria epidemiology and interventions in Ethiopia from 2001 to 2016. Infect Dis Poverty. 2018;7:1-9.

Gari

Lindtjørn

Reshaping the vector control strategy for malaria elimination in Ethiopia in the context of current evidence and new tools: opportunities and challenges. Malar J. 2018;17:454.

10.

Mohammed

Hong

Role of vector control in fighting against malaria: evidence from Ethiopian health-related indicators. J Infect Public Health. 2021;14:527-532.

11.

WHO. World Malaria Report 2022. World Health Organization; 2022.

12.

Durnez

Coosemans

. Residual transmission of malaria: an old issue for new approaches. In: Manguin

, ed. Anopheles Mosquitoes: New Insights into Malaria Vectors. IntechOpen; 2013:671-704.

13.

Debebe

Hill

Tekie

Ignell

Hopkins

RJ.

Shady business: understanding the spatial ecology of exophilic Anopheles mosquitoes. Malar J. 2018;17:9.

14.

Kenea

Balkew

Tekie

, et al. Human-biting activities of Anopheles species in south-central Ethiopia. Parasit Vectors. 2016;9:527.

15.

Yohannes

Boelee

Early biting rhythm in the Afro-tropical vector of malaria, Anopheles arabiensis, and challenges for its control in Ethiopia. Med Vet Entomol. 2012;26:103-105.

16.

Killeen

Govella

Lwetoijera

Okumu

FO.

Most outdoor malaria transmission by behaviourally-resistant Anopheles arabiensis is mediated by mosquitoes that have previously been inside houses. Malar J. 2016;15:225.

17.

Machani

Ochomo

Amimo

, et al. Resting behaviour of malaria vectors in highland and lowland sites of western Kenya: implication on malaria vector control measures. PLoS One. 2020;15:e0224718.

18.

Killeen

Chitnis

Potential causes and consequences of behavioural resilience and resistance in malaria vector populations a mathematical modeling analysis. Malar J. 2014;14:1-16.

19.

Yalew

AW.

Achievements, gaps, and emerging challenges in controlling malaria in Ethiopia. Front Trop Dis. 2022;2:771030.

20.

Sougoufara

Ottih

Tripet

FJ.

The need for new vector control approaches targeting outdoor biting anopheline malaria vector communities. Parasit Vectors. 2020;13:1-15.

21.

Bayoh

Walker

Kosgei

, et al. Persistently high estimates of late night, indoor exposure to malaria vectors despite high coverage of insecticide treated nets. Parasit Vectors. 2014;7:1-13.

22.

Perugini

Guelbeogo

Calzetta

, et al. Behavioural plasticity of Anopheles coluzzii and Anopheles arabiensis undermines LLIN community protective effect in a Sudanese-savannah village in Burkina Faso. Parasit Vectors. 2020;13:277.

23.

Bedasso

Gutto

Waldetensai

, et al. Malaria vector feeding, peak biting time and resting place preference behaviors in line with indoor based intervention tools and its implication: scenario from selected sentinel sites of Ethiopia. Heliyon. 2022;8:8.

24.

Lelisa

Hailemeskel

Bekele

Dugassa

Malaria positivity rate trend analysis at water resources development project of Wonji Sugar Estate Oromia, Ethiopia. Parasitol. Res. 2023;122:2259-2266.

25.

Tufa

Review on status, opportunities and challenges of irrigation practices in Awash River Basin, Ethiopia. Agrotechnology. 2021;10:1-10.

26.

WHO. Manual on Practical Entomology in Malaria: Methods and Techniques, Vol. 2. 1975.

27.

Silver

Mosquito Ecology Field Sampling Methods. Springer; 2008:1500.

28.

Massebo

Balkew

Gebre-Michael

Lindtjorn

Entomologic inoculation rates of Anopheles arabiensis in southwestern Ethiopia. Am J Trop Med Hyg. 2013;89:466-473.

29.

Vernoene

GA.

Outline for the determination of malarial mosquitoes in Ethiopia: Part I-Adult female

Anopheles Mosq News. 1962;22:1-13.

30.

Gillies

MT.

Studies of house leaving and outside resting of Anopheles gambiae Giles and Anopheles funestus Giles in East Africa I: the outside resting population. Bull Entom Res. 1954;45:375-387.

31.

Detinova

. With special reference to some vectors of malaria age-grouping methods in diptera with special reference to some vectors of malaria. In: Beklemishiv

, ed. Monogr Series of World Health Organization, Vol. 47. 1962:1-213.

32.

Haileselassie

Zemene

Lee

, et al. The effect of irrigation on malaria vector bionomics and transmission intensity in western Ethiopia. Parasit Vectors. 2021;14:516.

33.

Githeko

Service

Mhogo

Atieli

Resting behaviour, ecology and genetics of malaria vectors in large scale agricultural areas of western Kenya. Parassitologia. 1997;38:481-89.

34.

Msugupakulya

Kaindoa

Ngowo

, et al. Preferred resting surfaces of dominant malaria vectors inside different house types in rural south-eastern Tanzania. Malar J. 2020;19:22.

35.

Kibret

Lautze

Boelee

McCartney

How does an Ethiopian dam increase malaria? Entomological determinants around the Koka reservoir. Trop Med Int Health. 2012;17:1320-1328.

36.

Tsegaye

Demissew

Hawaria

, et al. Susceptibility of primary, secondary and suspected vectors to Plasmodium vivax and Plasmodium falciparum infection in Ethiopia. Parasit Vectors. 2022;15:384.

37.

Hawaria

Kibret

Demissew

, et al. Survivorship of Anopheles gambiae sensu lato in irrigated sugarcane plantation scheme in Ethiopia. Parasit Vectors. 2021;14:142.

38.

Adugna

Getu

Yewhelew

Parous rate and longevity of anophelines mosquitoes in bure district, northwestern Ethiopia. PLoS One. 2022;17:e0263295.

39.

Taye

Seid

Gindaba

Entomological study on species composition, behavior, longevity and probability of surviving sporogony of Anopheles mosquitoes in Lare District, Ethiopia. Parasitol Vector Biol. 2017;9:137-145.

40.

Gari

Kenea

Loha

, et al. Malaria incidence and entomological findings in an area targeted for a cluster-randomized controlled trial to prevent malaria in Ethiopia: results from a pilot study. Malar J. 2016;15:145.

41.

Kibret

Wilson

Tekie

Petros

Increased malaria transmission around irrigation schemes in Ethiopia and the potential of canal water management for malaria vector control. Malar J. 2014;13:360.

42.

Edalat

Moosa-Kazemi

Abolghasemi

Khairandish

Determination of age and vectorial capacity of Anopheles Maculipennis Sensu Lato in the Central Plateau of Iran. J Entomol Zool Stud. 2015;3:9.

43.

Zollner

Ponsa

Garman

, et al. Population dynamics of sporogony for Plasmodium vivax parasites from western Thailand developing within three species of colonized Anopheles mosquitoes. Malar J. 2006;5:68.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

7.45 MB