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Genetic diversity studies provide important details on target trait availability and its variability, for the success of breeding programs. In this study, GBS approach was used to reveal a new structuration of genetic diversity and population structure of pigeonpea in Benin. We used a total of 688 high-quality Single Nucleotide Polymorphism markers for a total of 44 pigeonpea genotypes. The distribution of SNP markers on the 11 chromosomes ranged from 14 on chromosome 5 to 133 on chromosome 2. The Polymorphism Information Content and gene diversity values were 0.30 and 0.34 respectively. The analysis of population structure revealed four clear subpopulations. The Weighted Neighbor Joining tree agreed with structure analyses by grouping the 44 genotypes into four clusters. The PCoA revealed that genotypes from subpopulations 1, 2 and 3 intermixed among themselves. The Analysis of Molecular Variance showed 7% of the total variation among genotypes while the rest of variation (93%) was within genotypes from subpopulations indicating a high gene exchange (Nm = 7.13) and low genetic differentiation (PhiPT = 0.07) between subpopulations. Subpopulation 2 presented the highest mean values of number of different alleles (Na = 1.57), number of loci with private alleles (Pa = 0.11) and the percentage of polymorphic loci (P = 57.12%). We discuss our findings and demonstrate how the genetic diversity and the population structure of this specie can be used through the Genome Wide Association Studies and Marker-Assisted Selection to enhance genetic gain in pigeonpea breeding programs in Benin.

Detarium microcarpum Guill. & Perr. is a priority food tree species in West Africa, but its use pattern and conservation is little known across different sociocultural areas of Benin. In this study, we analyse the determinants of D. microcarpum traditional uses pattern and how these determinants influence the species’ conservation in Benin. Thus, 730 respondents were participated in semi-structure interview across the North and Central regions of Benin. The information related to local names, traditional uses of different plant parts, management systems and conservation of D. microcarpum was recorded from the respondents. Use value (UV), ethnobotanical use value (EUV) and organ use value (OUV) were calculated. These statistics were used to assess the structure and variability of traditional use categories of plant parts among the sociocultural groups, gender and age groups. Results indicated that local names were spatially structured and linked to local communities’ cultural origins, according to the recent human migration roads in West Africa. In total, 42 traditional uses of seven categories were gathered. UV in relation to sociocultural groups ranges from 7.62 (Idatcha and Fon) to 16.08 (Peulh and Lokpa); within the gender, UV ranges from 9.96 (women) to 11.15 (men); and within the age groups, UV ranges from 9.37 [18–30 years old] to 14.14 [65–100]. The species’ UV significantly depended on respondents’ sociocultural group, age and gender. Moreover, the age, gender and sociocultural groups significantly influenced the species’ use pattern. Ethnobotanical use value ranges from 0.35 (fodder) to 6.22 (traditional human medicine). OUV ranges from 2.62% (flower) to 42.38% (leaf). The various uses of the species’ roots, leaves and bark in traditional pharmacology and their high-quality firewood and tasty fruits determined the various local management systems. Thus, considering the current threats (intensive use of the roots, trunk, leaves and branches, and habitat degradation) conservation measures are needed to ensure the survival, conservation of distribution pattern and sustainable use of the species.

Background Insecticide resistance amongst vector populations is a major challenge, exacerbated by the continued use of the same active ingredients. The present study assessed the impact of long-lasting insecticidal nets (LLINs) bi-treated with chlorfenapyr-alphacypermethrin (PY-CFP LLIN) or pyriproxyfen-alphacypermethrin (PY-PPF LLIN) on the genetic structure of resistant populations of Anopheles gambiae in 60 clusters divided into three arms from three districts in southern Benin. Methods The study was conducted between September 2019 and October 2021 in 123 villages grouped in 60 clusters. Mosquitoes were collected indoors and outdoors using human landing catches (HLCs) in 4 households in each cluster every 3 months. After morphological identification, a subsample of An. gambiae sensu lato ( s.l.) was analysed by PCR to detect the molecular species and the presence of L1014F vgsc-kdr and G119S -ace-1 mutations. Results Anopheles coluzzii (56.9%) and An. gambiae sensu stricto ( s.s .) (42.8%), with a few hybrids (0.2%), were identified within 4242 samples of An. gambiae tested. The frequency of L1014F vgsc-kdr decreased in An. coluzzii collected both indoors and outdoors locations in the PY-CFP LLIN and PY-PPF LLIN arms post-intervention compared to baseline. In An. gambiae , the frequency of the L1014F allele decreased in year one but increased above baseline in year 2. In both species, the allelic frequency of G119S -ace-1 was < 10%. For L1014F vgsc-kdr , the fixation index was positive ( F IS  > 0) in both species. However, it was negative ( F IS  < 0) for the presence of G119S -ace-1 . Weak genetic differentiation, especially in the PY-PPF LLIN and PY-CFP LLIN arms ( F ST  ≤ 0.05), was observed in An. gambiae s.s. populations with L1014F vgsc-kdr , while it was generally higher for both species with G119S -ace-1 . Conclusion The frequency of the L1014F vgsc-kdr resistance allele was high, while that of the G119S- ace-1 allele was low throughout the study period. Consistent changes in allele frequencies were not observed in any of the treatment arms suggesting that the pyrethroid component of dual AI (active ingredients) nets continues to select for the resistant allele and there is little if any evidence that the non-pyrethroid insecticide selects for the wild-type kdr allele.

Selection of mosquito collection methods is of crucial importance to evaluate the impact of vector control tools on entomological outcomes. During a cluster randomised control trial evaluating the relative efficacy of two dual-active ingredient (a.i.) long-lasting insecticidal nets (LLINs) compared to pyrethroid-only LLINs, we assessed the performance of different mosquito collection methods: Human landing catches (HLC), Centers for Disease Control and Prevention (CDC) light traps, and pyrethrum spray catches (PSC). Anopheles mosquitoes were collected using three collection methods in 4 houses, in each of the 60 trial clusters at baseline and every quarter for 24 months using PSCs and HLCs, while CDC light traps were performed during two quarters only. Mean density of vectors collected per method per night was the highest with HLCs (15.9), followed by CDC light traps (6.8); with PSCs (1.1) collecting 10 times less mosquitoes than HLCs. All three collection methods collected fewer mosquitoes in the Interceptor G2 ® dual a.i. arm, compared to the other trial arms, although only HLCs and PSCs demonstrated strong evidence of this due to a greater number of collection rounds undertaken, than CDC light traps. The broadly similar results regarding the differential impact of the two dual a.i. LLINs showed by the three collection methods suggest that the more ethically acceptable, cheaper, and logistically simpler methods such as CDC light traps could be prioritised for use in large community trials for measuring the efficacy of vector control tools.

Background Insecticide resistance is threatening the effectiveness of efforts to control malaria vectors in Benin. This study explores the levels and mechanisms of insecticide resistance in An. gambiae s.l. to pyrethroids. Methods Larvae were collected from August 2017 to July 2018 in five communes in southern Benin (Adjohoun, Allada, Bohicon, Cotonou, and Porto-Novo) representing diverse ecological regions, and were reared in Benin’s insectary. Two- to five-day-old female mosquitoes from each district were exposed to multiple doses of deltamethrin and permethrin (1×, 2×, 5×, and 10×) using the WHO insecticide resistance intensity bioassay. The effect of pre-exposure to the synergist, piperonyl butoxide (PBO), was also tested at different pyrethroid doses. Molecular allele frequencies of kdr (1014F) and ace-1 R (119S) insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled from each study area. Results An. gambiae s.l. were resistant to pyrethroid-only exposure up to 10× the diagnostic doses in all the study sites for both deltamethrin and permethrin. Mortality was significantly higher in An. gambiae s.l. pre-exposed to PBO followed by exposure to deltamethrin or permethrin compared to mosquitoes exposed to deltamethrin or permethrin only ( p  < 0.001). The difference in mortality between deltamethrin only and PBO plus deltamethrin was the smallest in Cotonou (16–64%) and the greatest in Bohicon (12–93%). The mortality difference between permethrin only and PBO plus permethrin was the smallest in Cotonou (44–75%) and the greatest in Bohicon (22–72%). In all the study sites, the kdr resistance allele (1014F) frequency was high (75–100%), while the ace-1 resistance allele (G119S) frequency was low (0–3%). Analysis of the metabolic enzymatic activity of An. gambiae s.l. showed overexpression of nonspecific esterases and glutathione S-transferases (GST) in all study sites. In contrast to the PBO results, oxidase expression was low and was similar to the susceptible An. gambiae s.s. Kisumu strain in all sites. Conclusion There is high-intensity resistance to pyrethroids in southern Benin. However, pre-exposure to PBO significantly increased susceptibility to the pyrethroids in the different An. gambiae s.l. populations sampled. The use of PBO insecticide-treated bed nets may help maintain the gains in An. gambiae (s.l.) control in southern Benin. Graphical Abstract

Background African pangolins are currently experiencing unprecedented levels of harvesting, feeding both local demands and the illegal international trade. So far, the lack of knowledge on the population genetics of African pangolins has hampered any attempts at assessing their demographic status and tracing their trade at the local scale. We conducted a pioneer study on the genetic tracing of the African pangolin trade in the Dahomey Gap (DG). We sequenced and genotyped 189 white-bellied pangolins from 18 forests and 12 wildlife markets using one mitochondrial fragment and 20 microsatellite loci. Results Tree-based assignment procedure showed that the pangolin trade is endemic to the DG region, as it was strictly fed by the the Dahomey Gap lineage (DGL). DGL populations were characterized by low levels of genetic diversity, an overall absence of equilibrium, important inbreeding levels, and lack of geographic structure. We identified a 92–98% decline in DGL effective population size 200–500 ya—concomitant with major political transformations along the ‘Slave Coast’—leading to contemporaneous estimates being inferior to minimum viable population size (< 500). Genetic tracing suggested that wildlife markets from the DG sourced pangolins through the entire DGL range. Our loci provided the necessary power to distinguish among all the genotyped pangolins, tracing the dispatch of a same individual on the markets and within local communities. We developed an approach combining rarefaction analysis of private allele frequencies with cross-validation of observed data that traced five traded pangolins to their forest origin, c. 200–300 km away from the markets. Conclusions Although the genetic toolkit that we designed from traditional markers can prove helpful to trace the illegal trade in pangolins, our tracing ability was limited by the lack of population structure within the DGL. Given the deleterious combination of genetic, demographic, and trade-related factors affecting DGL populations, the conservation status of white-bellied pangolins in the DG should be urgently re-evaluated.

The present study aimed to assess mosquito species diversity, distribution, and ecological preferences in the Covè, Ouinhi, and Zangnanado communes, Southern Benin. Such information is critical to understand mosquito bio-ecology and to focus control efforts in high-risk areas for vector-borne diseases. Mosquito collections occurred quarterly in 60 clusters between June 2020 and April 2021, using human landing catches. In addition to the seasonal mosquito abundance, Shannon's diversity, Simpson, and Pielou's equitability indices were also evaluated to assess mosquito diversity. Ecological niche models were developed with MaxEnt using environmental variables to assess species distribution. Overall, mosquito density was higher in the wet season than in the dry season in all communes. A significantly higher Shannon's diversity index was also observed in the wet season than in the dry seasons in all communes (p < 0.05). Habitat suitability of An. gambiae s.s. , An. coluzzii, Cx. quinquefasciatus and Ma. africana was highly influenced by slope, isothermality, site aspect, elevation, and precipitation seasonality in both wet and dry seasons. Overall, depending on the season, the ecological preferences of the four main mosquito species were variable across study communes. This emphasizes the impact of environmental conditions on mosquito species distribution. Moreover, mosquito populations were found to be more diverse in the wet season compared to the dry season.

Background Long-lasting insecticidal nets (LLINs) may have different impacts on distinct mosquito vector species. We assessed the efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs on the density of Anopheles gambiae s.s. and An. coluzzii compared to pyrethroid-only nets in a three-arm cluster randomised control trial in Benin . Methods Indoor and outdoor collections of adult mosquitoes took place in 60 clusters using human landing catches at baseline and every 3 months for 2 years. After morphological identification, around 15% of randomly selected samples of An. gambiae s.l. were dissected to determine parity, species (using PCR). Results Overall, a total of 46,613 mosquito specimens were collected at baseline and 259,250 in the eight quarterly collections post-net distribution. Post-net distribution, approximately 70% of the specimens of An. gambiae s.l. speciated were An. coluzzii , while the rest were mostly composed of An. gambiae s.s. with a small proportion (< 1%) of hybrids ( An. gambiae/coluzzii ). There was no evidence of a significant reduction in vector density indoors in either primary vector species [ An. coluzzii : DR (density ratio) = 0.62 (95% CI 0.21–1.77), p  = 0.3683 for the pyrethroid-pyriproxyfen LLIN and DR = 0.56 (95% CI 0.19–1.62), p  = 0.2866 for the pyrethroid-chlorfenapyr LLIN, An. gambiae s.s.: DR = 0.52 (95% CI 0.18–1.46), p  = 0.2192 for the pyrethroid-pyriproxyfen LLIN and DR = 0.53 (95% CI 0.19–1.46), p  = 0.2222 for the pyrethroid-chlorfenapyr]. The same trend was observed outdoors. Parity rates of An. gambiae s.l. were also similar across study arms. Conclusions Compared with pyrethroid-only LLINs, pyrethroid-chlorfenapyr LLINs and pyrethroid-pyriproxyfen LLINs performed similarly against the two primary mosquito species An. gambiae s.s. and An. coluzzii in Benin. Graphical Abstract

Background To help with planning malaria vector control in Benin, the National Malaria Control Program launched a study to update the distribution of major malaria vectors and their role in Plasmodium falciparum transmission. The study aimed to go beyond the standard entomological inoculation rate (EIR) by incorporating the average sporozoite load of the mosquitoes. This is because the parasite load is a key factor in a successful infection. The research proposed combining the average P. falciparum sporozoite load with EIR to better determine the vectors’ true contribution to malaria transmission. Methods The study was conducted across 18 communes in Benin. Within each commune, two villages were chosen for mosquito collection using human landing catches (HLC) and pyrethrum spray catches (PSC). Heads and thoraxes from female Anopheles gambiae s.l. and Anopheles funestus mosquitoes were analyzed for the Plasmodium falciparum circumsporozoite antigen using the ELISA/CSP method. The corresponding carcasses were used for species identification via PCR. The P. falciparum sporozoite load was quantified in CSP ELISA-positive mosquitoes using the NZYTech real-time PCR kit. The contribution of vectors to P. falciparum transmission was first estimated by considering both their infection and bite rates. Subsequently, the relative contribution to transmission was further assessed by correlating the P. falciparum sporozoite load of the primary vectors with EIR. Results Anopheles coluzzii is responsible for 63.01% of malaria transmission, with an EIR of 87.7 infecting bites per person per year. This is followed by Anopheles gambiae, which accounts for 36.7% of transmission and has an EIR of 51.1 infecting bites per person per year. The contribution of Anopheles funestus is 0.24%. The study found that An. gambiae carries a higher load of Plasmodium falciparum sporozoites than An. coluzzii . Specifically, approximately 30% of An. gambiae individuals carried more than 10,000 sporozoites in their salivary glands, while less than 10% of An. coluzzii individuals had a comparable load. Conclusion This study clarifies the true contribution of malaria vectors to Plasmodium falciparum transmission by linking sporozoite load to the EIR. The findings will allow for a more accurate assessment of the vectors’ role in P. falciparum transmission in Benin.

Background Spices have always been used for their flavor-enhancement characteristics and for their medicinal properties. In Benin, scientific research on spices is scarce, despite their importance in the local population’s daily needs. This study investigated the diversity of wild spices and documented the associated traditional knowledge that can be used for their valuation, domestication, and sustainable management in the Sudano-Guinean Zone of Benin. Methods Data were collected during field expeditions using semi-structured interviews in ten localities across the three phytodistricts of the zone. Species richness and Shannon’s diversity index were estimated using species accumulation curves. Use report (UR), cultural importance, use value (UV) index, and informant consensus factor ( F ic ) were used to assess traditional knowledge on wild species, their local importance, and informants’ agreement among sociolinguistic groups. Priority wild spices were finally identified using an approach combining eight criteria (native status, economic value, ethnobotanical value, global distribution, national distribution, in-situ and ex-situ conservation status, legislation, and threats assessment) in four prioritization methods (point scoring procedure, point scoring procedure with weighting, compound ranking system, and binomial ranking system). Results A total of 14 species, belonging to 12 genera and 9 families, were inventoried. The most prominent families were Zingiberaceae (21.43%), Annonaceae (21.43%), and Rutaceae (14.29%). More than 200 specific uses were reported, with the Tchabè people holding the greatest level of knowledge (70 uses; UR = 5.70 ± 0.33). The culturally most important spices differed among sociolinguistic groups. Most of the informants agree on the use of the species among ( F ic = 0.72–0.98) and across the considered use categories ( F ic = 0.88–0.99). The highest UV were registered for Aframomum alboviolaceum (UV = 0.93), Lippia multiflora (UV = 0.76), and Aframomum angustifolium (UV = 0.18). Overall, people perceived wild spices as declining due to agriculture, grazing, and drought. Five species, A. alboviolaceum , L. multiflora , Monodora tenuifolia , Xylopia aethiopica , and Z. zanthoxyloides , were the most prioritized for conservation. Conclusions This study provides information relevant for the implementation of conservation and domestication actions of wild spices in Benin. Priority species could be integrated into traditional agroforestry systems (e.g., home gardens). However, for this to be effective, further research should be undertaken on morphological and genetic diversity and propagation methods of priority wild spices.