Ethical clearance was obtained from the Cameroon National Ethics Committee, Yaoundé (REF: N° 2022/12/1506/CE/CNERSH/SP) and administrative clearance from the Delegation of Public Health, Littoral Region (Re: DD20/L/MSP/DRSPL/CDS) after approval of the protocol. Special consideration was taken to minimize any health risks to the participants and involvement was strictly voluntary. The objectives of the study were explained in detail to each participant who signed a consent form. The participant’s documents were given a code to protect the integrity of the study subjects. At the end of the study, the participants were treated with doxycycline (200mg daily for 28 days).

The study was carried out in the Yabassi health district (Yabassi Sub-Division), situated in the Nkam Division in the Littoral Region of Cameroon. The study site in the equatorial rain forest lies between latitudes 4°27’16” N and longitudes 9°58’56” E ( Figure 1 ). It experiences an equatorial climate with a long rainy season (April to November) and a short dry season (December to March) with an average annual temperature of 26.8°C. Moderate temperatures range from 23-25°C and mean annual rainfall of 2-4mm. The average annual relative humidity is 80.8%. The main geographical feature is the Nkam River valley, flanked on both sides by a gently undulating plain with an average altitude of 1000m above sea level. The total population was estimated at 12,000 inhabitants, who are mainly farmers (small cash crop plantations of cocoa, palm oil plantains, bananas) and traders, while some are involved in hunting and fishing activities.

Culicoides species were collected using the drop trap technique (18, 19). Microfilarial (mf)-positive volunteers (≥ 21-year-old males) were assessed by thick blood smear technique. The volunteers (two per session, having ≥520mf/ml of blood) were placed under a rectangular netting cage trap (3x2x2x2m), which was raised for 15- 20 min to allow contact between the host and midges and then lowered to trap the attracted midges. After about 15-20 mins (the expected time for most of the trapped night-biting midges to be fully blood-fed), the engorged Culicoides species trapped on the netting materials were gently aspirated by a well-trained fly collector technician with a locally made mechanical aspirator and blown into 50 ml Falcon tubes (Merck KGaA, Darmstadt, Germany), 3/4 filled with plaster of Paris (POP). Using a bright torch light, fully fed midges were separated from (aspirated) non-engorged or partially engorged midges into a different tube and labeled. Fully engorged midges could be seen from the blood-filled reddish abdomen. The POP formed an absorbent layer at the bottom of the tubes to retain moisture. Biting midge collections were performed between 6 p.m. and 6 a.m. and caught midges were maintained in the tubes for 3-5 days in the field as previously described (23). The collected biting midges were fed with 15% sugar solution and then transported to the main research laboratory in Manjo in a cool box for maintenance.

Culicoides species were maintained at 24°C and relative humidity (RH) of 75 ± 5% in the insectarium at the Manjo laboratory ( Figure 2 ). In short, a hole (0.5 cm in diameter) was made in the lid of each rearing tube to permit ventilation and feeding of the midges in captivity. This hole was covered with fine netting (pore size 0.5 mm) on which the sugar solution-moistened cotton was placed for midge-feeding. Engorged Culicoides were fed daily with 15% sugar solution soaked in cotton gauze. Additionally, 3–4 drops of distilled water were added daily using a 10 ml syringe to keep the bottom of the tubes moist. To record midge survival, the rearing tubes were inclined and slightly agitated. Generally, insects have the tendency of moving upward, especially in a closed system. With this in mind, we could easily determine motile and immotile midges. The immotile midges were considered dead after about 10-20 seconds of observation. Dead midges were removed from the tubes daily and stored in 80% alcohol. Mortality of the midges was recorded daily and immobile midges were declared dead after observing them for one minute.

Morphological identification was done on euthanized engorged Culicoides species following the examination of the wing pigmentation pattern and male genitalia under a dissecting microscope using a combination of identification keys (24, 25).

After a total of 12 and 14 days of maintenance in the insectarium, the live Culicoides species were knocked down in tween 20-killing solution for 1-2 minutes and rinsed in distilled water for approximately 15-30 seconds. The dead midges were placed on sterile microscope slides containing a drop of the dissecting medium, RPMI-1640 or DMEM (Sigma-Aldrich, Munich, Germany) supplemented with a 2% antibiotic cocktail (penicillin-streptomycin-neomycin; Thermo Fisher Scientific, Schwerte, Germany).

Under the dissecting microscope (Motic, Wetzlar, Germany), the head, thorax and abdomen of the knocked down midges were separated and teased just after morphological identification. This set-up allowed for 1-2 minutes for the larvae to migrate out of the various anatomical regions. All infective larvae (L3) were isolated into sterile dissecting wells labeled head (H), thorax (T) and abdomen (A) containing the same medium.

Data were collected daily on record sheets and entered into a template designed on Microsoft Office Excel 2007 (Microsoft, Redmond, USA). Analyses were performed using R Statistical Language version 4.4.0 (26). Descriptive analysis was conducted to illustrate the number of collected midges per collection season, and per month, as well as the number of live and dead midges dissected, the numbers of recovered L3 from dissected midges per donor and sampling date and Culicoides body part (head, thorax, and abdomen). The ggplot2 (27) package was used for data visualization. A Mann-Kendall Trend test was used to determine whether the trend was monotonic in time series data (mean daily collection of midges at different months, monthly dissection of C. milnei, C. grahamii and others). The association between the donor’s load and midge survival was investigated by fitting a Cox proportional hazard regression model (also referred to as Cox regression in this document) with “time to death” as the outcome variable and the donor’s microfilaria load as a predictor variable using the function coxph from the package survival 3.7-0 (28, 29). The model was then used to determine the predicted survival probability, and visualized with Kaplan Meier curves using the package survminer 0.4.9 (30). The number of recovered L3 produced by infected live midges was compared among donors using the Kruskal-Wallis rank sum test, followed by Dunn’s multiple comparison test using rank sums with Bonferroni adjustment when the former test revealed a significant difference (p-value<0.05).

A total of 13,658 engorged Culicoides (midges) were collected from February 2022 to January 2023. Five Culicoides species were identified namely; C. milnei, C. grahami, C. inornatipennis, C. fulvithorax and C. neavei as anthropophilic night biters. The mean parasite load of the six different donors, their respective ages and the period of midge collection are shown in Table 1 . Two donors were used per session and the mean parasite load of the donors were quantified every 3 months throughout the study period.

The months with the highest number of days of midge collection were March 2022 and October 2022 with 14 days of collection each, while during the months of December 2022 and January 2023 only 3 days of collection each was performed. In total, 13,658 midges were collected within 112 days between February 2022 and January 2023 ( Table 2 ). In general, two donors per week always participated in the midge collection

The average daily collection of engorged Culicoides was analyzed by calculating the number of engorged midges collected during a month divided by the number of days of collection of the respective month. In general, we grouped the months into seasons ( Figure 3 ) and differentiated them into the early dry season (mid-October, November, early December), peak dry season (mid-December, January, February, early March), early rainy season (mid-March, April, early May) and peak rainy season (mid-May, June, July, August, September, early October). February to May (early and peak dry season) showed the lowest values of average daily collection of midges with a weak and gradual increase (33.45 in February, 35.57 in March, 45.44 in April, and 51.50 in May). The increase became more evident from June to October (152.17 in June and 179.21 in October) with slight fluctuations (peak rainy season). The peak average daily midge collection was observed in December (457 midges per day), an early dry season month with low or no rainfall and moderate temperatures ( Table 2 ; Figure 3 ). After the early dry season, we noticed a drastic drop in collection in January which is the peak dry season. A Mann-Kendall trend test revealed the presence of a monotonic trend (p-value = 0.002), that is the direction of variation (increase here) in the daily average collection remained consistent throughout months of collection.

The average daily dissection of midges was higher throughout the year in C. milnei as compared to all other species combined ( Figure 4 ). C. milnei displayed a gradual increase in average daily dissection from the peak dry season to the rainy seasons (early and peak) with a drop in September. From the peak rainy season to the early dry season, there is a sharp rise in December (400 midges/day) followed by a drastic drop in January (150 midges per day, the peak dry season. Mann-Kendall trend test revealed a consistent increase in the daily average dissection of midges through months for C. milnei (p-value = 0.003), while other species did not demonstrate a monotonic variation (p-value = 0.45). The trend in other species combined is similar, but we noticed a drop in the number of midges dissected per day during the peak rainy season. There was no monotonic evolution (Mann-Kendall trend test, tau = 0.18, p=0.45) of the average daily dissection of C. grahamii and other midges combined ( Figure 4 ).

Culicoides collection and isolation of infective larvae from dissected live and dead midges differs between the six donors. To decipher whether a high microfilaria load is associated with dead midges, we dissected dead midges mainly from late July 2022 to January 2023 (dead midges from donor AA were not analyzed). Donor FF (who participated in the study from May 2022 to January 2023) recorded the highest number of engorged midges (n=7,294) caught and dissected both alive (n=3,931) and dead (n=619), followed by donor DD with 4,021 engorged Culicoides collected from which 2,432 alive and 144 dead midges were obtained ( Figure 7 ). We collected (136) and dissected (101 live midges and 4 dead midges) the least from donor BB (March to May 2022).

Out of the five engorged live Culicoides species (C. milnei, C. grahamii, C. inornatipennis, C. fulvithorax and C. neavei) identified from the donors after 12 and 14 days of laboratory maintenance, three were common in all six donors but C. neavei was only obtained from donor DD and EE ( Figure 8A ). C. milnei was the most abundant species across all the donors accounting for more than 50% of midges identified and dissected for every donor. C. milnei accounted the highest for donor DD (94.74%) followed by 93.16% for donor FF for all midges dissected. Overall, C. milnei contributed 87.23% of all live midges dissected in this study ( Figure 8A ). In contrast, only four Culicoides species ( Figure 8B ) were dissected dead from the donors (C. neavei absent). C. milnei accounted for more than 97.13% of all dead midges dissected of four out of five donors (donor BB, 25%) with the highest from donor FF (98.87%) followed by 97.22% from donor DD.

C. milnei accounted for 98.7% (n=6,310 L3) of total L3 recovered, while for dead dissected midges, 99.92% L3 recovery was obtained from the same species ( Figure 9A ). Throughout the dissection of live and dead midges ( Figure 9B ), L3 was recovered more from the head (62.54% and 66.11% in live and dead midges, respectively), followed by thorax (28.29% and 28.9% in live and dead midges, respectively) and lastly by abdomen (9.18% and 4.99% in live and dead midges, respectively). The donor FF (16,540mf/ml) with the highest microfilaria load contributed the highest to L3 production (83.65%) followed by donor DD (7,640mf/ml) with 9.05% during dissection of live midges. This was similar to results from the dissection of dead midges in which donor FF still contributed the highest to L3 recovery of 94.48% ( Figure 9C ).

The overall L3/infected midges from the head, thorax and abdomen (combined) for donors AA, BB, CC, DD and EE was significantly different for midges that fed on donor FF as compared to all other donors (Kruskal-Wallis, P<2.2e-16, Figure 10A ). In the head, a significant difference of recovered L3 from infected midges was found between donor FF and all other donors, except donor EE (Kruskal-Wallis, P<2.2e-16, Figure 10B ). The L3 from the thorax of infected midges from various donors varied significantly (Kruskal-Wallis, p=1.3e-07, Figure 10C ) and the source of this variation was identified between the pairs BB-FF and DD-FF. The L3/infected midge from the abdomen showed no significant difference (Kruskal-Wallis, p=0.53) amongst the donors. No L3 was recovered from the abdomen of live midges that were fed on donor AA and BB. The highest L3 recovered from infected midges was on donor FF for all anatomical regions (head, thorax and abdomen). An overview of live and dead midges and collected L3 from the different donors and body parts of the midges are shown in Tables 3 and 4 , respectively. Moreover, a spreadsheet about individual results from the midges is shown in the Supplementary Material .

As depicted in Table 3 , out of the 13,658 midges that were collected, a total of 8,123 Culicoides survived the rearing and were dissected over a period of 12 months (February 2022-January 2023). In total, we recovered 6,310 L3 from 2,335 M. perstans-infected midges, resulting in 2.70 L3 per infected midge. A total of 7,086 dissected C. milnei were responsible for 98.7% (6,228 L3) of total L3 recovered. Only 58 L3 were obtained from 690 C. grahamii dissections. In addition, 12 L3 each were obtained from 199 C. inornatipennis and 145 C. fulvithorax. C. neavei (3 collected) were all negative for M. perstans L3 ( Table 3 ). In total, 459 L2 and no L1 were obtained during the dissection process ( Supplementary Table 1 ). The majority of infective larvae were obtained from the head region (n=3,946), followed by thorax (n=1,785) and the abdomen (n=579; Table 3 ). Moreover, we analyzed dead midges from the donors’ BB, CC, DD, EE, FF ( Table 4 ). In total, 800 dead midges have been analyzed during the collection months of February to April 2022 and June to November 2022. In total 1,322 L3 was recovered from all dead midges but 1,321 have been obtained solely from dead C. milnei midges and 1 L3 from C. grahamii ( Table 4 ). The graphical representation of various replicates and, detailed numbers about infected dead midges and L3s, dissected dead midges and recovered dead L3 from the anatomical regions are shown in Supplementary Figure 1 and Supplementary Tables 2–4 respectively. From dead midges dissected, we recorded 377 infected dead midges. A total of 1,249 dead L3 were obtained from donor FF, who had a high mircofilaria load. In contrast, only 32 dead L3 was obtained from 73 dead infected midges out of 181 dead midges dissected from donor BB, CC, DD and EE combined, who had lower microfilaria numbers in the periphery.