Background

Malaria threatens millions of lives globally. According to the world malaria report of 2020, an estimated 229 million cases and 409 thousand deaths occurred worldwide in 2019 [1]. Although there was a reduction in the number of malaria cases in 2019, compared to 2015, no significant progress was documented [1]. A substantial increase in malaria was documented in 2020 [2] due to the COVID-19 pandemic and population increases. Africa accounted for about 94% of global cases. This region is also known for the most notorious malaria vectors [3]. Ethiopia is one of the malaria-endemic countries and contributed a substantial number of malaria cases in 2019. Plasmodium falciparum and P. vivax are the two dominant parasites in Ethiopia [2].

In Ethiopia, malaria affects millions of people, particularly in the fertile lowland areas. Most parts of the country are favorable for malaria mosquitoes and parasite transmission. Malaria prevention and control strategies began in the 1950s as pilot projects in Ethiopia [4]. Since then, vector control has been a key preventive method for malaria. Currently, insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS) are the two main malaria intervention tools [5]. The effectiveness of these interventions, however, varies due to the vector species and their behavioral responses to intervention tools. There are spatial and temporary variations in the feeding and resting behaviors and the species composition of malaria mosquitoes. For example, the principal malaria vector in a particular area could be a secondary vector in the other and vice versa. This knowledge clearly indicates the importance of continuous surveillance and monitoring of malaria mosquitoes to inform control programs [6].

Entomological information, such as vector species composition, their role in parasite transmission, feeding, and resting behaviors is crucial in a vector control program. These data provide information about the intensity of malaria transmission and help in designing and implementing an appropriate intervention [6]. Moreover, information that is collected through entomological surveillance assists in understanding the effectiveness of malaria vector control interventions and the spatial and temporal changes in vector species [7]. The accurate identification of malaria mosquitoes is highly important in a vector control program. Morphological identification in this regard plays a key role in the immediate documentation of the most important malaria vectors and the implementation of control tools [8]. It is, however, more challenging in areas where several species cohabit, as well as in areas where morphologically similar species are present. A vector control program could be affected by misidentification of species in such areas.

We used PSC and CDC light trap techniques to investigate the Anopheles mosquito species composition, blood feeding patterns and their role in malaria transmission in two villages of Boreda district in Gamo zone, southwest Ethiopia. Molecular speciation of An. gambiae complex and An. funestus group was carried out. PCR was used to confirm those species that were morphologically closely related to either the An. gambiae complex or the An. funestus group using specific primers from the two Anopheles species complexes. This study also aimed to better understand the challenges posed by morphological misclassification in entomological monitoring and surveillance.

Materials and methods

Description of the study area

This study was conducted in the Kodo-Awsato Menuka and Zefine-Menuka Kebeles (= villages: the lower administrative level in Ethiopia) of Boreda district in Gamo zone (Fig. 1). Boreda district is found in Gamo zone, southwest Ethiopia. It is at a distance of 94 km from Arba Minch town, the capital of Gamo zone and is bordered with Wolaita Zone at the north, Mirab Abaya district at the south–east, Chencha district at the south–west and Kucha district at the west side of its location. The district has 31 villages and contains both highland and lowland areas. The 15 villages in the lowlands are malaria endemic. Kodo-Awsato Menuka and Zefine-Menuka villages are among the lowland villages of the district that are located about 16 km from Zefine town, the central town of the district. There were 270 households in Kodo-Awsato Menuka village and 390 in Zefine-Menuka village. The estimated population of Kodo-Awsato Menuka village is 1830 and it was 2680 in Zefine-Menuka village.

Fig. 1
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Map of the study area; SNNPR is including the new regional states

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Agriculture is the basic economic activity for the inhabitants of the two villages. The villages are malarious. This study was carried out in localities with high malaria transmission to generate entomological data which provide information on the local disease transmission pattern and dynamics.

Study design and mosquitoes sampling

Entomological sampling was done for 5 months from October 2019 to February 2020 using two entomological sampling techniques, such as pyrethrum spray and CDC light traps.

Pyrethrum spray collection

PSC was employed to sample indoor resting adult Anopheles mosquitoes in selected households. Five houses were selected randomly from each study village for the indoor resting mosquito collection. The selected households were visited twice monthly. Informed consent was obtained from all subjects and/or their legal guardian(s).

The indoor resting collection was carried out in the morning from 6:00 am to 8:00 am. KWiK insecticide all-purpose insect killer with striking powder (made in Kuwait; ingredients: pyrethroid with PBO) was used for spraying the houses to knock down the indoor resting mosquitoes. The openings of the roofs and eaves were closed by a piece of cloth to prevent the mosquitoes from escaping, and the food items in the room were removed and heavy food storages closed carefully before spraying the houses. Then, the floor and furniture were covered with a white sheet and the spray administered by two persons, one outside and the other inside the house, starting at one side and moving around the house in a circular manner. The house was kept closed for 10 min and the knocked-down mosquitoes were collected from the white sheet. The mosquitoes were then transferred to vials using forceps and preserved with silica gel before traveling to Arba Minch University Medical Entomology Laboratory for further analysis.

CDC light traps collection

Twenty houses, 10 from each village, were selected at random to collect indoor host-seeking mosquitoes using CDC light traps (Model 512, John W. Hock Company, Gainesville, Florida, USA). The CDC light traps were put in place 45 cm above the feet of a person who slept beneath an insecticide-treated mosquito net that was subsequently thoroughly cleaned. The traps were hung at 7:00 p.m. and collected at 7:00 a.m. the next morning. All other people in the trapping room were instructed to sleep under the insecticide net. The trap bags were tightened to prevent mosquitoes from escaping the bags. The mosquitoes were then transferred to vials and preserved with silica gel. The specimens were transported to Arba Minch University Medical Entomology Laboratory for further analysis.

Mosquito identification

The standard morphological key was used to identify female Anopheles mosquito species [9]. Female Anopheles mosquitoes were categorized as unfed, fed, gravid, or half-gravid using a microscope. The sibling species of An. gambiae complex [10] and An. funestus group [11] were further identified by PCR in the Arba Minch University Medical Entomology laboratory. Some species morphologically closely related to An. gambiae or An. funestus were tested using the primers of An. gambiae complex and An. funestus group. Anopheles gambiae complex, An. salbaii, An. dancalicus, An. maculipalpis and An. fuscivenosus were tested using primers of An. gambiae complexes, whereas the An. demeilloni and An. funestus group were tested using primers of An. funestus complexes. Legs and wings were used for the molecular identification of the Anopheles species.

CSPs detection

The female Anopheles mosquitoes were processed for CSPs detection in the Arba Minch University Medical Entomological laboratory. The heads and thoraces of all the collected female Anopheles mosquitoes were used for the detection of the CSPs of P. falciparum and P. vivax_210 malaria parasites using Enzyme-Linked Immuno-Sorbent Assay (ELISA) [12]. The heads and thoraces were homogenised in 50 μL of blocking buffer (BB) using a plastic grinder. Then, 100 μL of BB was added twice to bring the final volume to 250 μL per mosquito. The BB was removed from the plates after 1 h; 50 μL of each homogenized mosquito was added per plate. The P. falciparum and P. vivax_210 positive sample and laboratory-colony of An. arabiensis were used as negative controls, respectively. After 2 h incubation, the plates were washed twice with PBS-Tween 20. Then, Horseradish peroxidase (HRP)-conjugated monoclonal antibody was added to each plate, and after 1 h of incubation, the plates were washed 3 times with PBS-Tween 20. Finally, 100 μL of peroxidase substrate was added per well and incubated for 30 min. The plates were observed visually for a green color and their optical density was determined at 414 nm in the microplate ELISA reader. Samples with a green color and with optical density values of greater than two times the average optical density of the negative controls were considered as sporozoite-positive.

Blood–meal origin assay

Freshly fed Anopheles mosquitoes from CDC light traps and PSC were examined for blood–meal origins determination with human and bovine antibodies by the ELISA technique [13] in different microtiter well-plates. The abdomen of Anopheles mosquitoes was homogenized in 50 μL of phosphate-buffered saline (PBS) solution (pH 7.4) and diluted to a volume of 200 μL by PBS. Then, 50 μL of the sample was added to each well in a 96-well microtiter plate and incubated overnight at room temperature. PBS containing a Tween-20solution was used to twice-wash each well. Then, 50 μl of host-specific conjugate (anti-human IgG and anti-bovine IgG) was added in each well of separate 96-microtiter plates and incubated for 1 h. The wells were then washed three times by PBS-Tween-20 solution and finally 100μlof peroxidase was added to each well. After 30 min the absorbance of 405 nm was recorded with an ELISA plate reader (MRX Microplate Reader, Dynex Technologies, 20,151–1683, Woonsocket, Rhode Island, USA). Human and bovine blood meals were used as a positive control, and unfed laboratory reared An. arabiensis was used as a negative control. To record the results as positive, the absorbance value should exceed the mean plus three times the standard deviation of four negative controls.

Data analysis and interpretation

The data were assessed and presented based on the methods utilized in the data collection. All the data were entered and analyzed using SPSS version 20. The entomological inoculation rate and sporozoite rate were calculated. The sporozoite rate (SR) is the fraction of vector mosquitoes with Plasmodium sporozoite protein in their salivary glands. The entomological inoculation rate (EIR) is calculated as the product of the human-biting rate and the sporozoite rate [14]. The SR and human-biting rate (HBR) were determined for PSC catches. For PSC-based EIR, the standard EIR was calculated as (number of sporozoite-positive ELISAs/number of mosquitoes tested) × human-biting rate (HBR) (which is the number of freshly fed mosquitoes per total number of occupants in the collection room). A Chi-square test was carried out to compare the human blood index (HBI) and bovine blood index (BBI) of the Anopheles mosquitoes. This statistical analysis was done to assess the Anopheles mosquito's tendency for blood–meal sources.

Ethical approval

Informed consent was obtained from all household heads and/or their legal guardian(s). The study was conducted in accordance with the Declaration of Helsinki and the protocol was reviewed and approved by the Ethics Review Board of Arba Minch University (CMHS/12033594). Verbal consent was obtained from village and district authorities before carrying out the mosquito collection. The objective of the study was explained to all concerned bodies.

Results

Anopheles species composition

A total of 680 Anopheles mosquitoes were collected by CDC light traps (n = 550; 80.9%) and PSC (n = 130; 19.1%). Fourteen Anopheles mosquito species were morphologically identified in the study area (Table 1). Anopheles demeilloni was the predominantly species collected (61.7%), followed by An. arabiensis (28.8%).

Table 1 Morphologically identified Anopheles mosquitoes species collected by CDC light traps and PSC from villages in Boreda district, southwest Ethiopia (October 2019–February 2020)
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Molecular identification of Anopheles species

A total of 635 Anopheles mosquitoes were tested using the primers of either An. gambiae or An. funestus complexes among those with some morphological closeness. There was morphological misidentification of Anopheles species. Among the 420 that were morphologically identified as An. demeilloni and tested for An. funestus complex, 11 (2.6%) were An. leesoni, the species of the An. funestus complex. Of the six that were morphologically identified as An. salbaii and tested for An. gambiae complexes, four were An. arabiensis. Furthermore, An. arabiensis were morphologically misidentified as An. maculipalpis and An. fuscivenosus (Table 2).

Table 2 Molecular identification of Anopheles mosquitoes collected from Boreda district of Gamo zone, southwest Ethiopia (October 2019–February 2020)
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Of the 196 An. gambiae complex that were tested for sibling species, 183 (93.4%) were amplified for An. arabiensis; the rest were failed to amplify. Anopheles leesoni was the only An. funestus group identified in the area.

Plasmodium CSP rates

A total of 680 female Anopheles mosquitoes (190 An. arabiensis and 490 other species including unidentified specimens) were tested for CSPs, of which a single An. arabiensis was found to be positive for P. falciparum CSPs from PSC collection. The overall P. falciparum CSP rate of An. arabiensis was 0.53% (1/190: 95% CI 0.01–2.9).

Entomological inoculation rate

The EIR is obtained from the product of human-biting rate (HBR) (which is the number of freshly fed mosquitoes per total number of occupants in the room for PSC and the SR). The P. falciparum EIR of An. arabiensis was 1.5 ib/p/5 months.

Blood meal sources

A total of 145 fresh fed Anopheles mosquitoes, 124 from the CDC and 21 from PSC, comprising An. demeilloni, An. arabiensis, An. leesoni and unidentified specimens were tested to detect their blood meal origins. Among the Anopheles mosquitoes that were tested for blood meal origins, 84 (57.9%; 95% CI 49.5–66.1) had bovine blood origins. Only 22 (15.2%; 95% CI 9.8–22.1) fed on human blood meal alone and about 24 (16.5%; 95% CI 10.9–23.9) had mixed blood meal origins of both human and bovine (Table 3).

Table 3 Blood meal origins of freshly fed Anopheles mosquitoes collected from Boreda district of Gamo zone, southwest Ethiopia (October 2019–February 2020)
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Anopheles demeilloni predominantly fed on the bovine blood meal origins and showed statistical significance (Table 4). The bovine blood index (BBI) of An. demeilloni, including the mixed blood meal origins was 80.9 (95% CI 73.0–87.4); the human blood index (HBI), including mixed blood meals was 28.6 (95% CI 20.9–37.3). Anopheles arabiensis had blood meal sources of both human and bovine, though most of them had more human blood origins than bovine which was also statistically significant (Table 4). The HBI of An. arabiensis was 64.3% (95% CI 35.1–87.2), while the BBI was 14.3% (95% CI 1.8–42.8).

Table 4 Comparison of human (including mixed) and bovine blood meal origins (including mixed) of freshly fed An. demeilloni and An. arabiensis collected from Boreda district of Gamo zone, southwest Ethiopia (October 2019–February 2020)
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Discussion

About 14 Anopheles species were morphologically identified in the Boreda district of southwest Ethiopia. Though An. demeilloni was the dominant anopheline species, only An. arabiensis was found to be positive for P. falciparum CSPs, demonstrating that An. arabiensis continues to be an important vector of malaria in this region. Furthermore, the blood meal source analysis demonstrated that An. demeilloni prefers to feed on bovine sources, while An. arabiensis were more likely to have blood meals of human origin. There was morphologically misidentification of An. leesoni from An. funestus group and An. arabiensis from An. gambiae complex.

There was high species diversity of malaria mosquitoes in the study district. Unlike many other studies in the region [15,16,17], An. demeilloni was the dominant species in the current study villages. Though the dominance of An. arabiensis varied, its principal role in malaria transmission was the same as is documented in many other studies. ITNs and IRS have brought change in the species composition and dynamics of malaria mosquitoes in many malaria-endemic countries [18]; little or no change has been documented against An. arabiensis. After the long implementation of vector control tools, An. arabiensis has maintained its position as a primary vector of malaria in many malaria endemic countries [18]. Control of this species may demand more intervention tools in addition to the existing ones [19]. There might be an increased level of malaria transmission outdoors in the present day due to the changing behavior of malaria mosquitoes. The need to collect malaria mosquitoes outdoors is crucial to assess the risk of people working at night outdoors. Moreover, the predominance of An. demeilloni and the observed human–vector contact may require attention for further research to comprehend the role of this species.

From the total tested Anopheles mosquitoes for CSPs, An. arabiensis was found to be positive for P. falciparum CSP. The overall P. falciparum CSP rate of An. arabiensis was comparable with the CSP rate from Sille village in 2006 [15] and Chano Mille in 2013 [17]. However, there is a methodological difference; in the current study, entomological sampling was carried out by CDC light traps and PSC, while Taye et al. [15] was carried out by HLC. The collection method that was employed by Massebo et al. [17] was similar to the methods used in the current study. The findings of the current and previous studies have shown that An. arabiensis continues to play a primary role in malaria transmission in Ethiopia [16, 17, 20]. On the other hand, it is important to understand the variation in the CSP rate of An. arabiensis in different areas, and the method of collection [20]. We did not confirm the presence of the Plasmodium parasite by a PCR, nor did we expose it to heat to check for the heat stability of the antigen. Therefore, there could be the possibility of a false positive.

The EIR of An. arabiensis was calculated from the PSC collection. Accordingly, the P. falciparum EIR of An. arabiensis was 1.5ib/p/5 months, indicating the existence of active malaria transmission. There are a few studies who have attempted to estimate the EIR in Ethiopia by following similar mosquito sampling techniques [17, 21]. Therefore, it could be possible to compare the current result with other studies. This result is comparable with a study in south-central Ethiopia [21] and the southwest Rift Valley [17], and it implies that this species plays a key role in malaria transmission in the region. Future malaria control programs might focus on expanding the control toolbox to address the challenge that is related to An. arabiensis [22]. This species has opportunistic and flexible feeding and resting behaviors which could challenge future control programs [23].

The blood meal origins test result of the current study showed that most of An. demeilloni displayed the tendency to feed on bovine blood meal, while An. arabiensis showed the tendency to feed on human blood meal. Similar studies confirmed the tendency of An. demeilloni to feed on bovine blood meal [24]. In the current study, An. arabiensis exhibited a tendency to feed on human blood rather than bovine blood, somewhat contrasting other studies that documented the bovine blood feeding tendency of An. arabiensis [24, 25]. Several studies have indicated the opportunistic feeding behavior of An. arabiensis which is mainly determined by the accessibility of the hosts [26, 27].

There was a morphological misidentification of Anopheles mosquitoes. Species with some morphologically similar structures with An. gambiae or An. funestus complexes were molecularly identified using the primers of the two species. Among the An. salbaii, An. dancalicus, An. maculipalpis and An. fuscivenosus that were morphologically identified, a substantial number were An. arabiensis. Similarly, a substantial number of An. leesoni were morphologically identified as An. demeilloni. This implies that morphological identification alone may result in the wrong speciation of Anopheles mosquitoes due to the morphological similarity of some species, and this in turn might affect malaria control and elimination programs. Furthermore, morphological identification alone might contribute to the wrong documentation of Anopheles species. Though the molecular technique is more expensive and time consuming than a morphological technique, the correct identification of the species using molecular technique might benefit the control program [28]. Morphological misidentification of the species may occur when specimens have lost important external characteristics [8]. The knowledge of the entomology staff also has an impact on the morphological identification of the species. Thus, it is essential to advance entomological knowledge to minimize problems with misidentification.

This study has some drawbacks. Although PCR technology was used to correct the incorrect species identification of Anopheles, further sequencing utilising species-specific areas, such as ITS 2, is still necessary to validate the gene alignments, since various species may have genes amplified by species-specific primers. In addition, just a small number of Anopheles mosquitoes were examined for the source of their blood meals, and the statistical significance that resulted from this limited sample size might result in incorrect conclusions.

Conclusions and recommendations

The current study revealed the existence of the varieties of Anopheles species that were identified using morphological and molecular methods. The dominant Anopheles species was An. demeilloni, followed by An. arabiensis. Anopheles arabiensis was the only vector of malaria in the area that was found to be positive for P. falciparum CSPs. According to the current blood meal sources analysis, most of the tested Anopheles mosquitoes displayed the tendency to feed on bovine blood meal origin, and hence animal-based intervention could be recommended. Because the Anopheles species was incorrectly identified based on morphology, the correct identification of the Anopheles species may necessitate the use of molecular techniques.