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Background Vesicular stomatitis virus (VSV), a vector-borne pathogen of livestock, emerges periodically in the western US. In New Mexico (NM), US, most cases occur close to the Rio Grande River, implicating black flies ( Simulium spp.) as a possible vector. In 2020, VS cases were reported in NM from April to May, although total black fly abundance remained high until September. We investigated the hypothesis that transience of local VSV transmission results from transient abundance of key, competent black fly species. Additionally, we investigated whether irrigation canals in southern NM support a different community of black flies than the main river. Lastly, to gain insight into the source of local black flies, in 2023 we collected black fly larvae prior to the release of water into the Rio Grande River channel. Methods We randomly sub-sampled adult black flies collected along the Rio Grande during and after the 2020 VSV outbreak. We also collected black fly adults along the river in 2021 and 2022 and at southern NM farms and irrigation canals in 2022. Black fly larvae were collected from dams in the area in 2023. All collections were counted, and individual specimens were subjected to molecular barcoding for species identification. Results DNA barcoding of adult black flies detected four species in 2020: Simulium meridionale  ( N = 158),  S. mediovittatum ( N  = 83),  S. robynae  ( N = 26) and S. griseum/notatum  ( N = 1). Simulium robynae was only detected during the VSV outbreak period, S. meridionale showed higher relative abundance, but lower absolute abundance, during the outbreak than post-outbreak period, and S. mediovittatum was rare during the outbreak period but predominated later in the summer. In 2022, relative abundance of black fly species did not differ significantly between the Rio Grande sites and farm and irrigation canals. Intriguingly, 63 larval black flies comprised 56% Simulium vittatum , 43% S. argus and 1% S. encisoi species that were either extremely rare or not detected in previous adult collections. Conclusions Our results suggest that S. robynae and S. meridionale could be shaping patterns of VSV transmission in southern NM. Thus, field studies of the source of these species as well as vector competence studies are warranted. Graphical Abstract

Background Entomological data for onchocerciasis surveillance relies on sampling black flies through human landing collectors in the field and laboratory testing of the flies for infection using pooled screening O-150 PCR-ELISA assay. Both techniques require improvements. This study aimed to optimize the Esperanza Window Trap (EWT) for black fly collection. We tested alternative carbon dioxide (CO 2 ) mimics to attract black flies to the traps. Additionally, we evaluated new quantitative PCR (qPCR) methods that target mitochondrial DNA markers and have been proposed to enhance the sensitivity and specificity for detecting Onchocerca volvulus infections in blackflies. Methods Traps baited with low, medium and high release rates of either 2-butanone or cyclopentanone as CO 2 mimics were field tested against traps baited with organically generated CO 2 in four ecological zones in Nigeria: Guinea savannah, derived savannah, rainforest and montane forest. The performance of EWTs baited with CO 2 or in combination with 2-butanone (low release) were subsequently evaluated against the human landing collection (HLC). Trap scaling was also pilot tested by comparing two EWTs to a single HLC team. Collected black flies were used to test detection of O. volvulus in black flies using Ov ND5 real-time PCR (qPCR) compared to the conventional pool screening O-150 PCR. Results EWTs baited with 2-butanone caught similar numbers of black flies ( Simulium damnosum s.l.) to those baited with CO 2 , while cyclopentanone collected significantly fewer flies in all locations. The low release of 2-butanone was the most effective overall, although HLCs collected higher numbers of black flies than EWT baited with CO 2 either singly or in combination with low-release 2-butanone. The combination of two EWTs baited with CO 2 and deployed 100 m apart from each other collected similar numbers of flies as one HLC. More black fly pools were positive for O. volvulus by Ov ND5 qPCR compared with O-150 PCR in derived savannah (31.15 vs. 15.57%), montane forest (11.54 vs. 0%) and rainforest (23.08 vs. 2.56%), with only one positive pool in Guinea savannah detected by both methods. Conclusions The 2-butanone has potential to be used in xenomonitoring as a standardized replacement for organically generated CO 2 . Ov ND5 qPCR detected more positive pools than O-150 PCR. The positive pools found in foci hitherto considered to have interrupted/eliminated onchocerciasis highlight the need for more sensitive and specific methods that support programmatic assessments to identify and combat recrudescence. Graphical Abstract

BACKGROUND: Simulium damnosum s.l., the most important vector of onchocerciasis in Africa, is a complex of sibling species that have been described on the basis of differences in their larval polytene chromosomes. These (cyto) species differ in their geographical distributions, ecologies and epidemiological roles. In Ghana, distributional changes have been recorded as a consequence of vector control and environmental change (e.g. deforestation), with potential disease consequences. We review the distribution of cytospecies in southern Ghana and report changes observed with reference to historical data collated from 1971 to 2005 and new identifications made between 2006 and 2011. METHODS/RESULTS: Larvae were collected from riverine breeding sites, fixed in Carnoy’s solution and chromosome preparations made. Cytotaxonomic identifications from 1,232 samples (including 49 new samples) were analysed. We report long-term stability in cytospecies distribution in the rivers Afram, Akrum, Pawnpawn and Pru. For the rivers Oda, Ofin and Tano we describe (for the first time) patterns of distribution. We could not detect cytospecies composition changes in the upper Pra, and the lower Pra seems to have been stable. The elimination of the Djodji form of S. sanctipauli in the Volta Region seems to have had no long-term effects on the distribution of the other cytospecies, despite an initial surge by S. yahense. There has been a recent increase in the occurrence of savannah cytospecies in the river Asukawkaw, and this might be related to continuing deforestation. CONCLUSIONS: Cytospecies’ distributions have not been stable from 1971 to 2011. Although there are no obvious causes for the temporary appearance and subsequent disappearance of cytospecies in a particular location, a major influence has been vector control and migration patterns, probably explaining observed changes on the Black Volta and lower Volta rivers. Deforestation was previously implicated in an increase of savannah cytospecies in southern Ghana (1975–1997). Our data had little power to support (or refute) suggestions of a continuing increase, except in the Asukawkaw river basin.

Background The onchocerciasis focus surrounding the lower Mbam and Sanaga rivers, where Onchocerca volvulus is transmitted by Simulium damnosum s.l. (Diptera: Simuliidae), was historically the largest in the southern regions of Cameroon. Annual community-directed treatment with ivermectin (CDTI) has been taking place since 2000, but recent studies have shown that new infections are occurring in children. We aimed to investigate blackfly biting and O. volvulus transmission rates along the lower Mbam river 16 years after the formal onset of annual CDTI. Methods Black flies were collected for three consecutive days each month between July 2016 and June 2017 at two riverside villages and two inland sites situated 4.9 km and 7.9 km from the riverside. Specimens collected at each site were dissected on one of the three collection days each month to estimate parity rates and O. volvulus infection rates, while the remaining samples were preserved for pool screening. Results In total, 93,573 S. damnosum s.l. black flies were recorded biting humans and 9281 were dissected. Annual biting rates of up to 606,370 were estimated at the riverside, decreasing to 20,540 at 7.9 km, while, based on dissections, annual transmission potentials of up to 4488 were estimated at the riverside, decreasing to 102 and 0 at 4.9 km and 7.9 km, respectively. However, pool screening showed evidence of infection in black flies at the furthest distance from the river. Results of both methods demonstrated the percentage of infective flies to be relatively low (0.10–0.36%), but above the WHO threshold for interruption of transmission. In addition, a small number of larvae collected during the dry season revealed the presence of Simulium squamosum E. This is the first time S. squamosum E has been found east of Lake Volta in Ghana, but our material was chromosomally distinctive, and we call it S. squamosum E2. Conclusions Relatively low O. volvulus infection rates appear to be offset by extremely high densities of biting black flies which are sustaining transmission along the banks of the lower Mbam river. Graphical Abstract

By any measure, such as abundance, species diversity or geographic range, the Simulium ornatum species group is one of the most successful Palearctic taxa of black flies. To explore potential diversity in this group in the Tian Shan range of Central Asia, we focused on Kyrgyzstan, in which three nominal morphospecies have been recorded. Among our samples, we morphologically identified S. mesasiaticum Rubtsov and a second possible species tentatively identified as S. ferganicum Rubtsov. By analyzing banding patterns of the larval polytene chromosomes, we discovered two fixed inversions, two sex-linked rearrangements, and 19 autosomal rearrangements, including supernumerary B chromosomes. The chromosomal data indicate minimal diversity of only one or two species across the surveyed area of nearly 50,000 km2. Mitochondrial DNA (CO1) sequences fell into three distinct clusters, possibly representing separate species. The chromosomal, molecular, and morphological data indicate that Kyrgyz populations are unique within the S. ornatum group, but the data sets are not entirely congruent. Thus, reconciling data sets and assigning existing names is tentative. Simulium mesasiaticum is linked with undifferentiated sex chromosomes, one of the three CO1 clades, and higher elevations, whereas S. ferganicum is tenuously associated with differentiated sex chromosomes, a separate CO1 clade, and lower elevations. These associations leave one Kyrgyz larva, which is in a third CO1 clade, unlinked to a formal species name. Our analyses also indicate that S. ornatum Meigen sensu stricto, contrary to previous reports, does not occur in Kyrgyzstan and should be deleted from the country’s faunal list.

Background: High ongoing Onchocerca volvulus transmission was recently documented in Maridi County, South Sudan. To complement community-directed treatment with ivermectin (CDTI) as the main onchocerciasis control strategy, we initiated a community-based vector control method “slash and clear” at the Maridi dam, a Simulium damnosum s.l. breeding site, to reduce O. volvulus transmission. Methods: Simulium damnosum s.l. biting rates were collected before and during the twenty months following the “slash and clear” intervention using the human landing catches. Black flies were dissected to measure parity rates before and twelve months after the intervention. Larvae and pupae of S. damnosum s.l. were collected from the dam for morphological and chromosomal characterization to identify the cytospecies involved. Results: Biting rates of S. damnosum s.l. close to the Maridi dam spillway decreased by >90% post-“slash and clear” for more than six months. Twelve months after the “slash and clear” intervention, the reduction in biting rates was still at <50% (p = 0.0007). Parity rates reduced from 13% pre-“slash and clear” (November 2019) to 5.6% post-“slash and clear” (November 2020). Larvae collected from the dam were identified as Simulium sirbanum. Conclusion: The “slash and clear” method was found to be an effective and cheap community-based method to reduce black fly biting rates caused by S. sirbanum. When repeated at least annually together with a high CDTI coverage, this intervention has the potential to considerably accelerate onchocerciasis elimination.

A man-biting black fly species so far regarded as Simulium rufibasis Brunetti in Thailand was morphologically analyzed. It was found to be almost indistinguishable in the female from S. rufibasis sensu stricto but distinguishable in the male by the slender fore basitarsus and abdominal segments 2, 6, and 7 each with a pair of shiny dorsolateral patches, and in the pupa by the absence of spine-combs on abdominal segment 7 and terminal hooks on segment 9. It is described as a new species, Simulium tenebrosum. Taxonomic notes are given to separate it from all 11 known species of the S. rufibasis subgroup of the Simulium (Simulium) tuberosum species-group. An analysis of the COI gene sequences shows that this new species is distantly separated from the closely related species.

A macrogenomic investigation of a Holarctic clade of black flies-the Simulium cholodkovskii lineage-provided a platform to explore the implications of a unique, synapomorphic whole-arm interchange in the evolution of black flies. Nearly 60 structural rearrangements were discovered in the polytene complement of the lineage, including 15 common to all 138 analyzed individuals, relative to the central sequence for the entire subgenus Simulium. Three species were represented, of which two Palearctic entities (Simulium cholodkovskii and S. decimatum) were sympatric; an absence of hybrids confirmed their reproductive isolation. A third (Nearctic) entity had nonhomologous sex chromosomes, relative to the other species, and is considered a separate species, for which the name Simulium nigricoxum is revalidated. A cytophylogeny is inferred and indicates that the two Palearctic taxa are sister species and these, in turn, are the sister group of the Nearctic species. The rise of the S. cholodkovskii lineage encompassed complex chromosomal and genomic restructuring phenomena associated with speciation in black flies, viz. expression of one and the same rearrangement as polymorphic, fixed, or sex linked in different species; taxon-specific differentiation of sex chromosomes; and reciprocal translocation of chromosome arms. The translocation is hypothesized to have occurred early in male spermatogonia, with the translocated chromosomal complement being transmitted to the X- and Y-bearing sperm during spermatogenesis, resulting in alternate disjunction of viable F1 translocation heterozygotes and the eventual formation of more viable and selectable F2 translocation homozygous progeny. Of 11 or 12 independently derived whole-arm interchanges known in the family Simuliidae, at least six are associated with subsequent speciation events, suggesting a facilitating role of translocations in adaptive radiations. The findings are discussed in the context of potential structural and functional interactions for future genomic research.

I analyzed 221 black fly larvae of the Simulium arcticum complex from the Logan River, Logan Canyon, Utah, for larval sexual dimorphism, chromosomal inversions, morphology of the centromere band of chromosome IIL, presence of B chromosomes, and frequency of the IS-1 autosomal polymorphism. All 2275 larvae collected at the Logan River were of the S. arcticum complex with one exception, which was S. canadense. I found a significant association between larval color and sex, but I did not find such an association between larval head patterns and sex. Nearly all chromosomally analyzed larvae had standard, noninverted IIL chromosomes in both sexes. However, 70 of the 117 noninverted IIL chromosome males had the enhanced/thin (Ce Ct) centromeric dimorphism. Thus, there are at least two Y chromosomes in the Logan River population (Y0 and Y1). I also found four females and three males that had different chromosomal inversions at the base of chromosome IIL, but each of these was a singleton. Of the 120 males analyzed, six larvae had one acrocentric B chromosome and one larva had two acrocentric B chromosomes. None of the larvae at the Logan River was polymorphic for the IS-1 autosomal inversion. The presence of Ce Ct individuals that are also st/st for the IIL arm suggests that some of these larvae at the Logan River may be a new taxon. This observation is significant since some populations defined as Simulium brevicercum have only the IIL-st/st, Ce Ce genotype, while others have IIL-st/st larvae that have both Ce Ce and Ce Ct centromeric bands.

Development times of eggs, larvae and pupae of vectors of onchocerciasis (Simulium spp.) and of Onchocerca volvulus larvae within the adult females of the vectors decrease with increasing temperature. At and above 25°C, the parasite could reach its infective stage in less than 7 days when vectors could transmit after only two gonotrophic cycles. After incorporating exponential functions for vector development into a novel blackfly population model, it was predicted that fly numbers in Liberia and Ghana would peak at air temperatures of 29°C and 34°C, about 3°C and 7°C above current monthly averages, respectively; parous rates of forest flies (Liberia) would peak at 29°C and of savannah flies (Ghana) at 30°C. Small temperature increases (less than 2°C) might lead to changes in geographical distributions of different vector taxa. When the new model was linked to an existing framework for the population dynamics of onchocerciasis in humans and vectors, transmission rates and worm loads were projected to increase with temperature to at least 33°C. By contrast, analyses of field data on forest flies in Liberia and savannah flies in Ghana, in relation to regional climate change predictions, suggested, on the basis of simple regressions, that 13–41% decreases in fly numbers would be expected between the present and before 2040. Further research is needed to reconcile these conflicting conclusions.