Genetic diversity and phylogenetic relationships of tsetse flies of the palpalis group in Congo Brazzaville based on mitochondrial cox1 gene sequences.

BACKGROUND: Despite the morphological characterization established in the 1950s and 1960s, the identity of extant taxa that make up Glossina fuscipes (s.l.) in the Congo remains questionable. Previous claims of overlap between G. fuscipes (believed to be G. f. quanzensis) and G. palpalis palpalis around Brazzaville city further complicate the taxonomic status and population dynamics of the two taxa. This study aimed to determine the phylogenetic relationships between G. fuscipes (s.l.) and G. p. palpalis and to assess genetic variation among G. fuscipes (s.l.) populations in Congo Brazzaville. METHODS: We collected 263 G. fuscipes (s.l.) from northern and central regions, and 65 G. p. palpalis from southern part of the country. The mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using taxa-specific primer pairs. Sequence data were analyzed in DnaSP and Arlequin to assess the genetic diversity, differentiation and demographic history of G. fuscipes (s.l.) populations. RESULTS: The general BLAST analysis yielded a similarity of 99% for G. fuscipes (s.l.) and G. p. palpalis. BLASTn analysis for G. fuscipes (s.l.) showed > 98% identity with GenBank sequences for G. fuscipes (s.l.), with BEMB population showing 100% similarity with G. f. fuscipes. Glossina fuscipes (s.l.) populations showed high haplotype diversity (H = 46, Hd = 0.884), moderate nucleotide diversity ( = 0.012) and moderate (FST = 0.072) to high (FST = 0.152) genetic differentiation. Most of the genetic variation (89.73%) was maintained within populations. The mismatch analysis and neutrality tests indicated recent tsetse population expansions. CONCLUSIONS: Phylogenetic analysis revealed minor differences between G. fuscipes (s.l.) and G. p. palpalis. Genetic diversity of G. fuscipes (s.l.) was high in the populations sampled except one. Genetic differentiation ranged from moderate to high among subpopulations. There was a restricted gene flow between G. fuscipes (s.l.) populations in the north and central part of the country. Genetic signatures based on cox1 showed recent expansion and recovery of G. fuscipes (s.l.) populations from previous bottlenecks. To fully understand the species distribution limits, we recommend further studies involving a wider sampling scheme including the swampy Mossaka focus for G. fuscipes (s.l.) and the entire range of G. p. palpalis in South Congo.

Comparative diagnostic and analytical performance of PCR and LAMP-based trypanosome detection methods estimated using pooled whole tsetse flies and midguts.

Detection of trypanosomes that cause disease in human beings and livestock within their tsetse fly hosts is an essential component of vector and disease control programmes. Several molecular-based diagnostic tests have been developed for this purpose. Many of these tests, while sensitive, require analysis of trypanosome DNA extracted from single flies, or from pooled tsetse fly heads and amplified trypanosome DNA. In this study, we evaluated the relative analytical and diagnostic sensitivities of two PCR-based tests (ITS and TBR) and a Trypanozoon specific LAMP assay using pooled whole tsetse flies and midguts spiked with serially diluted procyclics of a laboratory strain of Trypanosoma brucei brucei (KETRI 3386). Test sensitivity was also evaluated using experimentally infected tsetse flies. The aim was to determine the most appropriate pooling strategy for whole tsetse and midguts. RIME-LAMP had the highest diagnostic sensitivity (100%) followed by TBR-PCR (95%) and ITS-PCR (50%) in detecting trypanosome DNA from pooled tsetse midguts. RIME-LAMP also had the best diagnostic specificity (75%) followed by ITS-PCR (68%) and TBR-PCR (50%). The relative detection limit determined by serial dilution of procyclics was below 10(-6) (equivalent to 1parasite/ml). Using TBR-PCR, ITS-PCR and RIME-LAMP, it was possible to detect trypanosome DNA in single flies or in pools of 2, 3, 4, 5, 10, or 15 flies/midguts. The proportion of positive pools declined by up to 60% when testing pools of 15 whole flies as opposed to testing pools of 5-10 flies. Additionally, it was possible to detect DNA in a single infected tsetse fly in the background of 4, 9, or 14 uninfected tsetse flies. Averaged across pool sizes and tsetse species, RIME-LAMP detected the highest proportion of positive pools in spiked whole tsetse and midguts (86.6% and 87.2%) followed by TBR-PCR (78. 6% and 79.2%) and ITS-PCR (34.3% and 40.2%). There were no significant differences between the proportions of positive pools detected in whole flies and midguts. We conclude that pooling of whole tsetse/midguts is an effective strategy to reduce hands-on-time and hence has potential application in large scale xenomonitoring to generate epidemiological data for decision making. RIME-LAMP offers the best diagnostic sensitivity and specificity on pooled tsetse midguts, thus demonstrating its superior diagnostic performance when compared with TBR-PCR and ITS-PCR. Using pools of whole tsetse or midguts as source of DNA does not have any significant effect on test results and is more representative of the field conditions where the proportion of flies with infected midguts tends to be higher than flies with infected salivary glands. Therefore to save time and minimize costs, pooling of whole tsetse flies is recommended.