An entomological study of onchocerciasis vectors, Simulium damnosum s.l., in Kinshasa, Democratic Republic of Congo

Background Onchocerciasis, a parasitic disease caused by the larial worm Onchocerca volvulus transmitted through the bite of Simulium (black ies), is a cause of global concern, with the African population being majorly affected. This study focused on the bite rates, bite cycle, and transmission potential of Simulium damnosum s.l. in two sites with river blindness outbreaks in Kinshasa, DRC: Gombe (S1) and Mont-Ngafula at Kimwenza (S2). From we captured adult female black ies near breeding sites along the Congo River at S1 and Lukaya Valley at S2. Collections using human baits at the two sites were conducted for ve days/month. A total of 6082 black ies of species Simulium squamosum (classied based on other entomological surveys) were captured during the study period. The daily cycle of aggression revealed two peaks: one between 8 and 9 a.m. and the other between 4 and 5 p.m. Low bite rates were observed between 11 a.m. and 1 p.m. The annual biting rate/person reached 13,463 in S1 and 23,638 in S2, with a total of 37,101 bites/person. The average daily biting rate, 37 ± 10 and 69 ± 23 bites/day/person in S1 and S2, respectively, did not differ signicantly (P = 0.8901). The high density of the host population can disrupt the transmission of vector-borne diseases by diluting the transmission indices. There was no evidence of onchocerciasis transmission at the study sites because of inadequate laboratory facilities in the DRC. Various larval supports have been identied: at Gombe: aquatic plants, plastic bags, dead leaves, and rocks; at Kimwenza: Ledermaniella ledermanii (the most abundant species at the site), plastic bags, articial waste, and aquatic plants. two per standard of capturing adult ies, landing collection [23]”. Test samples were collected over ve days, between 7 a.m. and 6 p.m., to identify sites high black y activity assess staff performance in capturing black ies. The actual was done for 24 weeks, over 12 months. The collections by human baits were simultaneously performed at the two sites, a.m. and 6 p.m. The two staff capturing black ies at least 500 The teams exchanged regularly, every eciency staff black ies. The cycle


Introduction
Onchocerciasis, also called "river blindness", is a parasitic disease caused by the larial worm Onchocerca volvulus transmitted through the bite of infected black ies of the genus Simulium that breed in fast-owing streams and rivers [1]. Michael et.al. (2020) reported WHO estimates for onchocerciasis: at least 25 million people are infected worldwide and 90 million people are at risk, with more than 99% of them living in Africa [2]. Therefore, onchocerciasis has been a public health problem for decades in the Democratic Republic of Congo (DRC) with all 26 provinces affected; in 2016, the estimated overall prevalence was 14.65 million people, including 12.22 and 1.03 million suffering from skin disease and loss of sight, respectively [3,4]. The city-province of Kinshasa, DRC, also suffers from this, despite various control programs established for more than twenty years: e.g., the African Programme for Onchocerciasis Control (APOC), National Programme for Onchocerciasis Control, and National Programme for Neglected Tropical Diseases Control (PNLMTN-CP) [3,4]. The disease epidemiology is de ned by the presence of black y breeding sites [1,5]. The larvae of this insect are aquatic and rheophilic: the larvae attach to submerged supports, in running water, at a shallow depth, for oxygen and food [6]. S.damnosum s.i. was rst identi ed in 1903 in Kinshasa by an English expedition from the Liverpool School of Tropical Medicine; however, only in 1926, Blacklock and his team demonstrated that onchocerciasis was transmitted by black ies [7][8][9]. Several onchocerciasis sites have been identi ed in Kinshasa since then [4]. Wanson, Henrard, and Peel claimed S.damnosum as the only known vector in 1945, in Kinshasa villages, located on the bank of the Congo river, along the rapids [4]. In 1944 and 1945, Wanson and Henrard studied the behavior of S.damnosum in Kinshasa and located larval deposits mainly in the rapids, downstream of the city [4]. Other larval sites were also detected: along the Nsele river, 60 km from Kinshasa, in the north of Kisundu; along tributaries of the Congo River, about 100 and 130 km upstream from Kinshasa (the Lu mi River); Mfuti River near Sabuka in Mong Ngafula; along tributaries of the Lukunga, along Ndjili, and several other small rivers around Kinshasa, including Lukaya, the Binza River in Kinsuka, etc. [4]. A vector control program with the aerial spraying of dichlorodiphenyltrichloroethane was organized from 1948 to 1952 [10,11]. Brown, in 1962, reported that S.damnosum did not reappear in Kinshasa for 10 years after the eradication campaign. Kinshasa remained unscathed for several years until the ies reemerged in 1984 [12]. Till now, only three sites of onchocerciasis have been explored in the city: Kinsuka-pêcheur, N'sele, and Mont-Ngafula at Kimwenza [4,[10][11][12][13]. Several authors have con rmed the presence of disease vectors in the three sites: S.damnosum, S.squamosum [14,15], and S.albivirgulatum have been identi ed in the sites [16].
A fourth site, Gombe, was reported after 1945, in the north of the capital along the Congo river but was never explored [4,17]. This study determines the presence of the vector, S.damnosum s.l., in Kinshasa, particularly in the sites Mont-Ngafula at Kimwenza and Gombe.

Description of the study environment
Kinshasa, the capital of the DRC, is a large city (area: 9,965 Km 2 ), with 24 municipalities and four districts, and an estimated population: 11 million (population density: ±3,600 inhabitants/Km 2 ) [18].
Kinshasa has a tropical savannah climate, equatorial in nature (hot and humid), with a rainy season lasting 8 months and a dry season lasting 4 months-a dry winter. The average annual temperature and precipitation are 25.3°C and 1,273.9 mm, respectively [18].
The dry season (tropical winter), June to September, is characterized by rare and low rainfall and lowest temperatures [18]. The sky is generally overcast and vegetation suffers from a lack of water [18]. During the rainy season (tropical summer), March to May, the precipitation is between 1000 and 1500 mm [18]. Generally, in Congo, it is hot to very hot all year round. Maximum temperatures are 30-35ºC during rainy periods (peaks at 40°C); the temperature rarely drops to below 20°C in the dry season [18].
Rivers of various sizes drain the plains of Kinshasa; they generally originate in the hills, ow from south to north, and empty into the Congo river [18,20]. Lakes (smaller waterbodies than rivers), such as Lac Ma Vallée and Lac Vert, are also located here [18,20]. Some of these waterbodies exhibit geographical characteristics that enable rapid waterfalls compatible with the survival of Simulium, particularly, the Lukaya river in rural Kimwenza and Congo river in the urban part of the bay of Gombe [18,20]  The sites were explored two days before the study to locate the roosts of pre-imaginal forms of black ies, identify their xation supports, and select the vector-human contact points. The best contact points were de ned as the two capture points of anthropophilic adult black ies for determining entomological indices (S1 and S2).

Capture and dissection of adult black ies
Human baits were used to capture adult black ies based on Le Berre's method [13,14,21]. Capture points at each study site were selected following Enyong's criteria, including, proximal to black y-breeding grounds (along the river), accessible throughout the year, located in shade, sheltered from the wind, and distal from regular human activity [22]. Four staff, two per site, were trained in standard methods of capturing adult ies, "human landing collection [23]". Test samples were collected over ve days, between 7 a.m. and 6 p.m., to identify sites with high black y activity and assess staff performance in capturing black ies. The actual collection was done ve times a month, for 24 weeks, over 12 months. The collections by human baits were simultaneously performed at the two sites, between 7 a.m. and 6 p.m. The two staff capturing black ies were at least 500 m apart. The teams were exchanged regularly, every hour, to minimize any potential bias relating to the e ciency of staff capturing black ies. The hourly capture rates were noted to determine the daily bite rate (the cycle of aggression) [14].

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The study was conducted in a randomly chosen 24-week period, from August 1, 2019, to July 31, 2020, including the two main seasons in Kinshasa: rainy and dry seasons. During the rainy season, the water levels in the rivers were high, and so were the black y populations. All black ies captured at S1 and S2 were analyzed.

Determination of entomological indices and data analysis
The cycle and level of aggressiveness, as well as parity and transmission potential, were used to determine entomological indices, following WHO guidelines [24]. The cycle of aggression refers to the daily biting pattern of black ies. From an epidemiological and entomological viewpoint, this criterion determines the time of the day when the risks of bites and transmission are the greatest [14]. The average number of ies captured per hour (estimated using Microsoft Excel) determines the cycle. The aggressiveness rate is represented by the daily biting rate (DBR; the number of black y bites/day), monthly biting rate (MBR; the average number of bites per person per month), or annual biting rate (ABR; the sum of MBRs calculated for 12 months) [14]. Determining parity requires dissecting the black ies to distinguish between nulliparous and parous black ies. The parous black ies were then opened up by cutting the abdomen and thorax in a drop of physiological water and carefully examined for O.volvulus larvae. The team carefully noted the number, stage of development (L1, L2, or L3), and location of the larvae. The black ies harboring developing larvae (L1, L2, or L3), whatever be their location, were considered to be infected, while those harboring infective larvae were categorized as infectious [14,25].
Larvae of the S.damnosum complex were identi ed morphologically by the presence of tubers and dorsal abdominal scales on the prothoracic prolegs [26]. These dissections were conducted in the laboratory of the Kinshasa School of Public Health (ESP).
To assess the degree of onchocerciasis transmission by black ies, we calculated the monthly transmission potential ( infectious black y. The hourly catch of black ies was recorded, and the black ies were preserved in absolute ethanol before analysis at the bioecology laboratory for preliminary morphology, according to dichotomous keys proposed by Crosskey [26], and physiological age [26]. Furthermore, the entomological data were recorded daily on collection cards. Consistency of the information declared and potential omissions were rst assessed by the eld collectors. Later, the whole team ( y catchers, supervisors, and main researchers) reviewed all the collection tools to identify possible inconsistencies. Two-sample t-tests with unequal variances were used to compare the mean values of study parameters at the two capture sites (S1 vs. S2) and capture period; the χ ² or Fisher's exact tests were used to compare the proportions of parous females at the two capture sites. P < 0.05 was considered signi cant. The data were analyzed using Excel ™ and SPSS 20 ™ Data Analysis Software -.

Entomological indices
The results of black y capture, dissections, and onchocerciasis transmission indices are summarized in Tables 1 and 2. A total of 6082 black ies were captured during the study period, 2207 at S1 and 3875 at S2. The density of female black ies was different between S1 and S2 but not statistically signi cant (P = 0.2198); the average bites per day and per person were 37 ± 10 and 69 ± 23, respectively ( Fig. 2 and Table 4).
The ABR reached 37,101 bites per person. Of all dissected black ies, none carried O. volvulus, and no molecular test was performed due to the lack of laboratories in Kinshasa. There is no evidence of onchocerciasis transmission at the study sites.
These supports were found in abundance around the rapids of the Lukaya Valley and along the Congo River, especially in places frequented by humans from the surrounding areas. The pre-imaginal population was more prominent on Ledermanniella ledermannii (the main support) > arti cial supports > plant remains.

Black y species at the capture sites
The identi ed black y species, S.Squamosum, belonging to the S.damnosum s.l. complex, was the same as in Kinshasa. Its morphological characteristics are the color of wing tufts (tufts of hair at the base of the wings) and relative coloring of antennae, procoxa, and prosternum (antenna parts) [25].

Entomological parameters
This study identi ed the following entomological parameters for onchocerciasis transmission: MBR, ABR, parity, black y infection rate (percentage of infected and infectious parous females), and transmission potential.
The average physiological age of female black ies was expressed against the proportion of parous females (Fig. 4). The parous/nulliparous ratio helped estimate the survival rate of black ies, which was directly linked to the risk of disease transmission. In S1 (Table 1) and S2 (Table 2), the dissection of 2,207 and 3875 female ies revealed an average parity rate of 43% (N = 952) and 28% (N = 1075), respectively.    However, when studying these parous females, we could not validate infection with O.volvulus larvae (regardless of the stage). We preserved the samples for a thorough laboratory examination (molecular test: pool screening) abroad since the necessary laboratory facility was unavailable at DRC. The MTPs were 0 at S1 and S2 during the study period, with no parous females carrying O.volvulus (Tables 1 and 2). The daily bite cycle was diurnal; two peaks were observed: a moderately major peak between 8 and 9 a.m. and a major peak between 4 and 5 p.m. (Figs. 5 ). The lowest biting rate was between 11 a.m. and 1 p.m. The morning peak may be because human activity took place near the larval breeding sites at that time (Fig. 5).
The average number of black ies captured per hour during the study period was higher in the morning (before noon) and evening. However, the hourly changes were not statistically signi cant; P > 0.05 (Table 3). Since the calculated P-values were > 0.05, the averages of none of the variables showed statistical signi cance (Table 4).  [27]. Considering the supports identi ed at S1 and S2, this study con rms the breeding of S.damnosum at these sites; the ies lay their eggs in the river rapids, where the larvae hatch and develop into adults within eight to twelve days [28]. The pre-imaginal forms (eggs, larvae, and nymphs) are all aquatic and strongly rheophilic [28]. After hatching, the young larvae either remain attached to aquatic supports or drift with the current [28]. They feed with their rigid mandibular soles, which they use to randomly catch particles suspended in running water, including the nutrients they need [28].

Black y species at capture sites
Morphological examination of the collected larvae and adult females revealed that they all belonged to the species S.squamosum of the S.damnosum s.l. complex; this was in coherence with results from previous surveys by Mansiangi (2014) [14] and Makenga (2015) [25] in Kinshasa. Henry et al. had also identi ed the vectors of O.volvulus to belong to the S.damnosum s.l. complex during the 1984 Kinsuka outbreak [13].
However, Traoré et al. reported species of the S.neavei complex to be the only vectors at the Sankuru basin in DRC from July 1998 to January 1999 [25]. The credibility of onchocerciasis control programs depends on thoroughly evaluating the effectiveness of treatments. The entomological evaluation includes verifying the effectiveness of the treatment during an outbreak, by estimating larval mortality and determining the bite rate and onchocerciasis transmission parameters [29]. All species reported (S.squamosum of S.damnosum s.l. complex) exhibit the phenomenon of "limitation", a factor that favors disease transmission [30][31][32][33][34]. "Limitation" refers to vectors being effective even at very low parasite densities [35]; the greater the number of micro lariae ingested, the lower the percentage that reach the hemocoel [32]. Flies infected with a low micro laria load have a greater chance of surviving and transmitting parasites [33,36]. The peritrophic membrane facilitates "limitation" by reducing the number of ingested micro lariae that reach the hemocoel. This percentage of reduction is lower in tropical forests [37,38] than in savannahs [31]. Therefore, Simulium sp. found in forests transmit more infectious larvae than those in savannahs.

Entomological parameters
This study shows a diurnal y bite cycle; two peaks, a minor peak between 8 and 9 a.m. and a major peak between 4 and 5 p.m. (Fig. 5), were observed. Our results con rm the conclusions of Henry et al. (1984) who observed the same two peaks in the Kinsuka study [13].
Mansiangi et al. also observed two peaks at Kinsuka: a major peak between 4 and 5 p.m. and minor peak between 9 and 10 a.m. [14].
Makenga et al. also observed two peaks: a major peak in the morning and minor peak at around 5 p.m. [27]. This difference may have resulted from an increased y activity when people were concentrated around the breeding sites, namely in the morning and at sunset, as in our case. Our results corroborate with those of Nascimento-Carvalho in Brazil or Homoxi, where the biting activity of female Simuliidae showed a bimodal pattern, with peaks in activities early morning (between 7 and 8.50 a.m.) and afternoon (between 4 and 5.50 p.m.) [39]. In Thirei, the biting activity was evenly distributed throughout the day [40]. Black ies were captured simultaneously in Homoxi and Thirei using systematic methods to compare the traditional capture method using human bait (Human Landing Catch, HLC) with HLC protected by MosqTent® [39].  [14]. Changes in ecological conditions may explain this difference. Besides, although the Lukaya River is a small tributary and offers few breeding sites, just like the Congo River at Gombe, the fast-owing freshwater may be conducive for black y production (larval and pupal stages) and support the main ecological characteristics of black ies.

Conclusion
This study concluded that larval supports and vectors for onchocerciasis exist at both sites, Mont-Ngafula in Kimwenza and Gombe. S.squamosum is responsible for disease transmission at these sites. However, entomological indices of onchocerciasis transmission were low at the target sites during the study period. The high density of the host population can disrupt the transmission of vector-borne diseases by diluting the transmission indices.
Following the results of this study, we recommend the following to the Ministry of Public Health through PNLMTN-CP: 1. To provide DRC with a laboratory capable of molecular testing (pool screening).
2. To organize an epidemiological and entomological assessment at the four sites in Kinshasa according to the WHO/APOC protocol to enable the PNLMTN-CP to demonstrate the elimination of onchocerciasis from Kinshasa and monitor its re-emergence at these sites. These sites, located in Kinshasa city, have high population densities that may dilute the entomological indices and disrupt onchocerciasis transmission.
3. To perform entomological monitoring in Kinshasa to study the black y biting rates. This will enable PNLMTN-CP to collect the data necessary to advocate vector control.
To the Scienti c Community, we recommend promoting research in the eld of medical entomology, in general, and onchocerciasis entomology, in particular, to support the Ministry of Health in research and entomological evaluations of onchocerciasis across the country.   Variation in the number of parous females at the two capture sites, Gombe and Kimwenza, from August 2019 to July 2020, in the cityprovince of Kinshasa.

Figure 4
Proportion of parous black ies captured at the two sites, Gombe and Kimwenza, from August 2019 to July 2020, in the city-province of Kinshasa.

Figure 5
Daily cycle of aggression of female black ies in Gombe and Kimwenza during the study period, August 2019 to July 2020. H, hour.

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