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Phlebotomine sandflies of Botswana: a taxonomic review and a faunistic update with the first record of genus Phlebotomus
Accepted Manuscript Title: Phlebotomine sandflies of Botswana: A taxonomic review and a faunistic update with the first record of genus Phlebotomus Author: Andreas Kruger ¨ PII: DOI: Reference:
S0001-706X(17)30061-X http://dx.doi.org/doi:10.1016/j.actatropica.2017.03.009 ACTROP 4235
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
17-1-2017 9-3-2017 10-3-2017
Please cite this article as: Krddotuger, A.,Phlebotomine sandflies of Botswana: A taxonomic review and a faunistic update with the first record of genus Phlebotomus, Acta Tropica (2017), http://dx.doi.org/10.1016/j.actatropica.2017.03.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Phlebotomine sandflies of Botswana: A taxonomic review and a faunistic
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update with the first record of genus Phlebotomus
3 Andreas Krüger
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20359 Hamburg, Germany, and Okavango Research Institute, University of Botswana, P. Bag
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285, Maun, Botswana
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E-Mail: [email protected]
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Department of Tropical Medicine, Bundeswehr Hospital Hamburg, Bernhard-Nocht-Str. 74,
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RUNNING TITLE
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Updated sandfly records in Botswana
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ABSTRACT
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The first records of phlebotomine sandflies from Botswana have been published only
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recently, comprising of four species of genus Sergentomyia. This update presents the first
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record of genus Phlebotomus, namely Ph. (Anaphlebotomus) rodhaini Parrot, which is also
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the first detection of a putative vector of leishmaniasis in Botswana. In addition, records of
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the Sergentomyia “bedfordi (Newstead) group” are reviewed, and the molecular taxonomy of
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all taxa known from Botswana is analysed based on three mitochondrial gene fragments
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(mtDNA). The presence of Se. congolensis (Bequaert and Walrveus) and Se. salisburiensis
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(Abonnenc) is confirmed, whereas the previously mentioned Se. caliginosa Davidson and
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unassigned specimens of the “bedfordi group” are proposed to belong to the tentatively
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named Se. bedfordi “Maun” form. The mtDNA analyses confirmed the species delimitations.
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For the first time, portions of the ND5 gene were used for the purpose of sandfly molecular
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taxonomy. This gene revealed a high inter-specific variability and may thus be applied as an
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alternative molecular marker for future studies.
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KEY WORDS
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Afrotropical, Phlebotominae, taxonomy, cytB, COI, ND5
1. Introduction
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Sandflies (Psychodidae: Phlebotominae) are haematophagous nematoceran Diptera that are, in
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contrast to mosquitoes, blackflies and biting midges, independent from aquatic environments,
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as their offspring develops strictly terrestrial, e.g. in animal burrows and shelters, rock cracks,
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or caves. In many regions of the world they are obligate vectors of kinetoplastid parasites of
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the genus Leishmania, the causative agents of leishmaniasis infection. Major human disease
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foci are found across Africa (WHO, 2010), with the exception of southern Africa, where there
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are only small and scattered foci known in Namibia (Noden and van der Colf 2013) and
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Zambia (Alvar et al. 2012).
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The knowledge on the Afrotropical sandfly fauna increased between 1951 and 1993 from 58
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to 147 species and subspecies (Kirk and Lewis, 1951; Seccombe et al., 1993). The
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zoogeographical southern African sub-region with the Rhodesian Highland, the Southeast
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Veldt and the Southwest Arid districts (Kirk and Lewis, 1951), covering Namibia, Botswana,
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Zimbabwe, South Africa, Lesotho, Swaziland, as well as parts of Angola, Zambia and
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Mozambique harbours about 51 species, 32 of which are endemic to southern Africa
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(Seccombe et al., 1993). Only recently the first records of phlebotomines from Botswana were
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reported (Krüger, 2015, 2016), namely Sergentomyia (Grassomyia) inermis (Theodor, 1938),
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Se. (Ser.) congolensis (Bequaert and Walravens, 1930), Se. (Ser.) caliginosa Davidson, 1987
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and Se. (Ser.) salisburiensis (Abonnenc, 1967), the latter three of which belong to the Se.
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“bedfordi (Newstead, 1914) group”. If based on small sample sizes, misinterpretations of
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morphological characters in these small-bodied and closely related species can easily happen
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due to damaged body parts or suboptimal preparation of the microscopic slides containing the
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structures of interest such as cibarial armature, pharynx and genitalia. Analyses of further
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samples, and comparison with the initial specimens revealed the presence of two additional
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taxa one of which replaces the erroneously recorded Se. caliginosa.
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Taking advantage of the availability of specimens of closely related species from South
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Africa, Namibia and Cameroon, data on the molecular taxonomy of Botswana sandflies were
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also retrieved by conducting DNA sequence comparisons of the mitochondrial genes for
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cytochrome b (cytB) and cytochrome c oxidase I (COI). The latter is the most commonly used
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region for DNA barcoding (Hebert et al., 2003) and has previously been analysed in Indian
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Sergentomyia (Pradeep Kumar et al., 2012), whereby cytB analyses have already been
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conducted on sandflies from Afghanistan and elsewhere (Krüger et al., 2011). In addition, the
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5’ portion of the mitochondrial gene for the NADH dehydrogenase subunit 5 (ND5) was
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analysed for the first time in order to evaluate its suitability for phlebotomine taxonomy (Ye
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et al., 2015).
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2. Material and methods
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Details on the study area, sampling data, trapping technique, morphological sample
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preparation and species identification of 28 specimens have been published recently (Krüger,
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2015). In brief, the study area was in the vicinity of Maun, northern Botswana, the centre of
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Ngamiland district. The region is very flat at an elevation of about 940 m above sea level, and
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characterised by the unique combination of an inland river delta, surrounded by savannah and
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semi-desert. The sampling device was a Standard CDC light-trap (Model 512;
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John W. Hock, Gainesville, FL), deployed 50 cm beside a termite hill at a fan height of 50
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cm, about 10-200 m off Thamalakane river. One collection site was in Sexaxa ward, in the
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premises of the Okavango Research Institute (ORI), the second site in a private property in
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Disaning ward. The trap was run from 5pm to 8.30am during seven trap nights.
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An additional nine specimens (5 males, 4 females), whose analysis is reported here, have the
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same details and were processed accordingly (Table 1).
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Each mounted specimen was documented photographically and identified using the
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morphological keys provided by Abonnenc (1972) and Davidson (1990), the latter from
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which the taxonomy and terminology used here for the Sergentomyia “bedfordi group” was
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adopted.
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For genomic DNA extraction, ethanol-preserved, unmounted body parts (thorax and proximal
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parts of the abdomen, legs, wings) of each sandfly of interest were dried for 3 hrs at 37°C, and
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subsequently processed with the DNeasy blood & tissue kit (Qiagen, Hilden, Germany),
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following Qiagen’s supplementary protocol for purification of total DNA from insects, using
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disposable microtube pestles for homogenization. For comparison, further specimens of
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closely related species from Afghanistan (Kondoz town, collected 2009; Krüger et al., 2011),
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Cameroon (Mokolo town, collected 2012, unpublished) and South Africa (KwaZulu Natal,
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collected 2015 by A. Latif, unpublished) were processed, i.e. Se. distincta (Theodor, 1933),
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Se. schwetzi (Adler, Theodor & Parrot, 1929), Se. (Gra.) dreyfussi ssp. turkestanica Theodor
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& Mesghali, 1964, Se. (Gra.) ghesquierei (Parrot, 1929), and Se. (Gra.) squamipleuris
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(Newstead, 1912).
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All PCRs were performed in a Biometra TProfessional Basis thermocycler (Biometra,
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Göttingen, Germany). The protocols for cytB and COI PCRs were as described previously
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(Ready et al., 1997, Krüger et al., 2011, Pradeep Kumar et al., 2012), using GoTaq G2 Hot
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Start and GoTaq G2 Flexi polymerase, respectively (Promega, Mannheim, Germany). The
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only modification was for cytB reactions a 40 sec annealing during the first five cycles
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(instead of only 30 sec; Ready et al., 1997). For ND5 amplification, an approximately 510
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base pair long fragment at the 5’ end of the ND5 coding region was chosen, as this was shown
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to be the most variable region of the entire sandfly mitochondrial genome (Ye et al., 2015).
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The following primers were used: forward N5-J-6579 (modified from Simon et al., 1994)
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TTCWCTYCAWCCTTGATC; reverse N5-6500 (modified from Birungi and Munstermann,
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2002) GCBGGYTTTTATTCWAAGGA. PCR amplifications were performed in 50 µl
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volumes containing 1 µl of DNA template, 9 µl Go Taq Flexi buffer (Promega, Mannheim,
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Germany), 3 µl 25 mM MgCl2, 0.2 pmol/µl of each primer, 0.3 µl 10 mM dNTP mix, and 1.5
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U of Go Taq Hot Start polymerase. The thermal cycler conditions were the same as for cytB
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PCR (see above).
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All reactions were run at 2.2% FlashGel cassette agarose gels (Lonza, Rockland, USA).
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Positive reactions were purified with the NucleoSpin Extract II kit (Macherey-Nagel, Düren,
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Germany) and commercially sequenced by direct forward and reverse sequencing (Seqlab,
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Göttingen, Germany). The respective sequences were deposited in Genbank under accession
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numbers KY451779-451798 (COI), KY451799-451811 (ND5), KY451812-451833 (cytB).
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The cytB (422 base pairs) and ND5 (491 bp) sequences were individually run against the
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BLAST tool of the publicly available NCBI Genbank in order to search for highly similar
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sequences. Further, they were aligned using Clustal Omega
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(http://www.ebi.ac.uk/Tools/msa/clustalo/) and analysed in MEGA7 (Kumar et al., 2016).
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Neighbour-Joining (NJ) analyses were computed using the Kimura 2-Parameter method.
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Bootstrap values were calculated to test the robustness of the resulting taxon ID trees and
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were obtained by conducting 1000 replicates.
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For COI barcoding, the obtained sequences (533 bp) were initially run against two databases,
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i) by using the BLAST tool, and ii) the specimen identification tool of BOLD4 (Barcode of
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Life Data systems v4 (http://v4.boldsystems.org/index.php). In order to visualize the
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barcoding results, COI sequences were aligned and analysed as described above, in this case
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together with Genbank entry JX105042 for Ph. rodhaini Parrot, 1930 and BOLD reference
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sequence BNITM095-12 for Se. (Gra.) dreyfussi ssp. turkestanica from Afghanistan (detailed
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specimen records and sequence information including traces files can be found within the
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BOLD Working Group 1.4 Initiative “Human Pathogens and Zoonoses”: “Ticks and
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Sandflies at BNITM Hamburg 2012”).
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In addition to the 28 previously investigated specimens, another nine were morphologically
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identified to species level. The new specimens belonged to five species, three of which had
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been reported previously (Table 1): Se. (Gra.) inermis (2 additional specimens), Se. (Ser.)
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congolensis (1 more specimen) and Se. (Ser.) salisburiensis (2 additional specimens). One
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specimen that was previously assigned to Se. (Ser.) caliginosa of the “bedfordi group”, six
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specimens that were previously treated as “bedfordi group” sensu lato, and three new
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specimens, turned out to belong to a new taxon tentatively named Se. bedfordi “Maun” form.
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A single female fly was unequivocally identified as Phlebotomus (Anaphlebotomus) rodhaini.
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Three of the recorded species belong to the Se. “bedfordi group” and are rather delicate to
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distinguish. Updated morphometric measurements are summarized in Table 2. For
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comparison, data for Se. distincta, as well as published data for other relevant species (see
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below) are listed, i.e. for Se. caliginosa, Se. formica Davidson, 1987 and Se. bedfordi s.str.
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The decision for (re-)assigning 10 specimens to the new “Maun” form of Se. bedfordi was
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based on the observation that both males and females showed character combinations close to
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or intermediate between Se. caliginosa and Se. formica.
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In females of the “Maun” form, the shape of the cibarial pigment plate resembles that of Se.
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caliginosa and, to a lesser degree, of Se. bedfordi s.str., being oblong with a variable median
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projection (Fig. 1). In addition, the posterior margins are strikingly ragged. However,
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intermediate or Formica-like characters are as follows: cibarial armature with 41-50 closely
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packed, long fine, monomorphic teeth [according to Davidson (1990) 45-65, versus 35-42 for
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Se. caliginosa; see Table 2]; median posterior margin of hard palate with tuberculate
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denticles, though small (smooth in Se. caliginosa); apical margin of hypopharynx with deep
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indentations (weakly undulating in Se. caliginosa).
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Males of the “Maun” form (n=3) of Se. bedfordi exhibited the following characters: cibarial
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armature with 16-23 small, irregular teeth (16-24 in Se. caliginosa, versus 30-42 in Se.
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formica); posterior margin of hard palate (Fig. 2A, B) with one to three rows of small
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tuberculate denticles (large tooth-like eruptions in Se. formica, a cibarial anterior row of
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spiculate denticles in Se. caliginosa); pigment plate absent (present in Se. formica);
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Morphometrics (n=2): length of A3 125-134 µm; Ascoid-3 21-28 µm; Ascoid-4 21-23 µm.
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Figure 2C shows the cibarial armature of a male Se. salisburiensis for comparison, with a
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rather concave posterior margin of the hard palate bearing large tooth-like eruptions.
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3.2. Molecular taxonomy
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All three mitochondrial DNA analyses unequivocally confirmed the morphological species
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delimitations (Fig. 3), with high bootstrap support for the species clusters highlighting low
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intra-specific variability. Furthermore, the NJ genealogies are to a large extent in agreement.
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In particular the Botswana members of the “bedfordi group”, i.e. the “Maun” form of Se.
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bedfordi, Se. congolensis and Se. salisburiensis form a cluster together with Se. distincta from
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Cameroon. Another cluster consists of Se. inermis from Botswana and the related species of
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the subgenus Grassomyia, i.e. Se. dreyfussi turkestanica, Se. ghesquierei and Se.
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squamipleuris, respectively. Phlebotomus rodhaini is consistently divergent from the other
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taxa. In the COI analysis (Fig. 3A) however, Se. schwetzi from Cameroon is placed within the
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“bedfordi group” cluster, which could be due to insufficient taxon coverage with regard to the
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“schwetzi group” of related species (Davidson, 1990). Regarding the newly introduced ND5
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analysis (Fig. 3C) it can be resumed that this gene region is highly variable between species,
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resulting in slightly higher inter-specific genetic distances than in the COI and cytB regions,
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as deduced from the branch length scale.
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High similarity BLAST searches (megablast) of cytB sequences revealed high identity scores
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for the reference specimens Se. dreyfussi turkestanica (100% with Se. dreyfussi turkestanica)
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and Se. schwetzi (98% with Se. schwetzi), as well as for Ph. rodhaini (98% with Ph. rodhaini
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from CAR). All other taxa had scores of ≦94%, indicating the absence of con-specific
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Genbank entries. Megablast searches of COI sequences yielded a 99% identity score for Se.
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schwetzi, whereas for Ph. rodhaini it was only 94% with another Ph. rodhaini, which
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contained numerous undetermined base positions (“N”). For all remaining taxa the identity
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scores only reached less than 93%, again indicating the absence of con-specific Genbank
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entries. The ND5 sequences showed only up to 80% identity on blastn searches, whereby
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Genbank at present only contained three sequences of three species of phlebotomine
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sandflies.
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4. Discussion
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199 The most relevant finding of this study is the first record for Botswana of a putative vector of
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zoonotic leishmaniasis, which is Ph. rodhaini. Elnaiem et al. (2011) incriminated this sandfly
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species as a possible vector of Leishmania donovani in Sudan. Phlebotomus rodhaini is
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widespread in sub-Saharan Africa including Namibia and South Africa, but was so far not
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recorded in Botswana, Zambia, and Mocambique (Davidson, 1981, Niang et al., 2011). The
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Botswana specimen, a gravid female, was collected at light next to a termite hill in the gallery
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forest about 10 m off Thamalakane river in Maun. These conditions resemble those described
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by Davidson (1981) for sampling sites in Namibia (Mashare, Kavango region) and South
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Africa.
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Concerning the Se. “bedfordi group” the species delimitation turned out to be challenging.
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Abonnenc (1972) tried to shed light into the confusing taxonomy of the group then
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comprising 12 entities. Davidson (1990) subsequently again revised the group, now
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comprising 20 species. As an example of the persistent problems he added the following
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comment to the female holotype of Se. bedfordi s.str.: “This specimen appears to be unique
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and may be aberrant.” Contrary to Davidson (1990), Seccombe et al. (1993) retained certain
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species as invalid under Se. bedfordi s.str., e.g. Se. congolensis and Se. distincta.
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In the light of considerable morphological overlaps between the taxa (see Table 2), and the
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modern approach of integrated morphological and molecular taxonomy it seems mandatory to
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revise the “bedfordi group” again through a comparison of DNA sequences of typical
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specimens when these become available. At least, typical Se. caliginosa and Se. formica
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should be examined, as the newly designated “Maun” form could not be assigned to either of
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the two on a morphologically basis, but which at least exhibited a clear molecular distance
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from other members of the “bedfordi group”. Only then it can be proven whether the “Maun”
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form constitutes a new species, or only a variant or part of a hybrid population of the before
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mentioned species. This could be supplemented by analysing at least one non-mitochondrial
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marker (Depaquit, 2014).
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Concerning the presence of species of the subgenus Grassomyia in South Africa there is a
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slight confusion in Abonnenc (1972). The main text listed Se. inermis for this country, but the
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respective distribution map only showed Se. squamipleuris in South Africa. The present study
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revealed the latter to be true, but according to Abonnenc (1967) it seams reasonable to assume
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both species to be present, with Botswana adding a second southern African record of Se.
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inermis.
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There is no obvious explanation why Botswana’s invertebrate fauna has received so little
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attention, placing the country as a “white spot” on many faunal maps (Krüger and Deckert,
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2016). By 2016, the only inventories publicly available were on butterflies, dragonflies,
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grasshoppers and true bugs, leaving most insect taxa seemingly as absent in the country,
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including Isoptera (termites) or sandflies (Republic of Botswana, 2015; Krüger, 2015; Krüger
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and Deckert, 2016).
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Acknowledgement
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Annett Michel is gratefully acknowledged for excellent technical assistance. Professor
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Abdalla Latif is thanked for giving specimens. Parts of this study with regard to Cameroon
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specimens were funded by the Volkswagen foundation.
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web.mpl.ird.fr/identiciels/phlebotomes/html/index.html (visited 27Oct 2016).
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Parrot, L., 1929. Sur un autre phlébotome nouveau du Congo Belge Phlebotomus ghesquierei
317
n. sp. Rev. Zool. Bot. afr. 18, 90-91.
318
13
Page 13 of 24
319
Parrot, L., 1930. Sur une coleection de phlébotomes du Congo Belge. Rev. Zool. Bot. afr. 19,
320
181-192.
321 Pradeep Kumar, N., Srinivasan, R., Jambulingam, P., 2012. DNA barcoding for identification
323
of sand flies (Diptera: Psychodidae) in India. Mol. Ecol. Resour. 12, 414-420. Doi:
324
10.1111/j.1755-0998.2012.03117.x.
cr
ip t
322
325
Ready, P.D., Day, J.C., de Souza, A.A., Rangel, E.F., Davies, C.R., 1997. Mitochondrial
327
DNA characterization of populations of Lutzomyia whitmani (Diptera: Psychodidae)
328
incriminated in the peri-domestic and silvatic transmission of Leishmania species in Brazil.
329
Bull. Entomol. Res. 87, 187-195. doi: https://doi.org/10.1017/S0007485300027346.
M
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us
326
330
Republic of Botswana, 2015. Fifth National Report to the Convention on Biological
332
Diversity. Available from: https://www.cbd.int/doc/world/bw/bw-nr-05-en.pdf (accessed 10
333
January 2016).
te
Ac ce p
334
d
331
335
Seccombe, A.K., Ready, P.D., Huddleston, L.M., 1993. A catalogue of Old World
336
phlebotomine sandflies (Diptera: Psychodidae, Phlebotominae). Occas. Pap. Syst. Entomol. 8,
337
1-57.
338 339
Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P., 1994. Evolution,
340
Weighting, and Phylogenetic Utility of Mitochondrial Gene Sequences and a Compilation of
341
Conserved Polymerase Chain Reaction Primers. Ann. Entomol. Soc. Am. 87(6), 651-701. doi:
342
https://doi.org/10.1093/aesa/87.6.651.
343
14
Page 14 of 24
344
Theodor, O., 1933. Some African sandflies. Bull. Ent. Res. 24, 537-547.
345 346
Theodor, O., 1938. On African sandflies III. Bull. Ent. Res. 29, 165-173.
348
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347 Theodor, O., Mesghali, A., 1964. On the Phlebotominae of Iran. J. Med. Ent. 1, 285-300.
350
cr
349 WHO, 2010. http://www.who.int/leishmaniasis/leishmaniasis_maps/en/.
us
351
Ye F., Liu T, King, S.D., You, P.. 2015. Mitochondrial genomes of two phlebotomine sand
353
flies, Phlebotomus chinensis and Phlebotomus papatasi (Diptera: Nematocera), the first
354
representatives from the family Psychodidae. Parasit. Vectors. 8, 472. doi: 10.1186/s13071-
355
015-1081-1.
M
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352
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15
Page 15 of 24
i cr us
Date
Maun, Disaning, Termite hill
15.-16. xii 2014
Maun, ORI, Termite hill
26.-27. xi 2014
1f
Congolensis
"Maun" form
2f (1f)
1f
1.-2. xii 2014
2f
3.-4. xii 2014
2f
1f
9.-10. xii 2014
5f, 1m
3f
13.-14.i 2015
1f, 1m
ce
Salisburiensis
1f
2f, 2m
pt
S19°53'59" E23°31'43"
10.-11. i 2015
Inermis
1f, 1m
ed
S19°57'58" E23°27'43"
Rodhaini
M
Location
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Table 1. Summary of sampling locations, dates and numbers of collected sandfly species. Bold numbers: sequenced specimens.
1f, 1m 1m
1m
1f 2f, 1m
1f
1f
1f
12f, 4m
6f, 1m
2f, 1m
7f, 3m
TOTAL
1
16
7
3
10
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Sub-TOTALS
16 Page 16 of 24
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Table 2. Morphometric measurements of Sergentomyia “bedfordi group” females from Botswana, with counting of the cibarial teeth, lengths of antennal segments A3, A4 and A5. For comparison, further members of the group, which are either mentioned in the text or used for analyses,
M
are listed. SE: standard error; n: number of specimens measured. All lengths measures are given in micrometer.
Species:
ed
Botswana
A4
bedfordi s.str.
bedfordi “Maun”
distincta
distincta
formica
caliginosa
holotype
Range (n)
30-38 (5)
29-37 (2)
41-50 (5)
17-24 (17)
20-30 (30*)
45-65 (30*)
35-42 (10*)
20
Mean (SE)
35 (1.72)
--
43.8 (1.85)
21.8 (0.54)
--
--
--
--
Range (n)
120-136 (5)
127
105-122 (4)
105-135 (5)
100-140 (30*)
95-125 (30*)
90-120 (10*)
165
Mean (SE)
128.2 (3.35)
--
112.5 (3.57)
116 (5)
--
--
--
--
Range (n)
70-77 (5)
79
65-70 (4)
65-80 (5)
59-79 (30*)
56-72 (30*)
55-70 (10*)
87
Ac
A3
ce
Character Cibarial teeth
salisburiensis
According to Davidson (1990):
pt
congolensis
Cameroon
17 Page 17 of 24
i cr 70-80 (5)
90
Mean (SE)
75.2 (1.59)
--
72 (2)
--
--
--
--
68-70 (3)
70-85 (5)
--
--
--
--
69.3 (0.67)
76 (4)
--
--
--
--
Ac
ce
pt
* includes holo- or lectotype.
us
Range (n)
67 (1.22)
an
--
M
73.2 (1.12)
ed
A5
Mean (SE)
18 Page 18 of 24
Fig. 1. Cibaria of female Sergentomyia bedfordi “Maun” form from Botswana. The dark
2
structures in the centre represent the pigment plates. Right panel: same specimen at different
3
focus levels, above for pigment plate, below for cibarial armature with 41 teeth.
4
(Nomarsky/DIC microscopy).
ip t
1
5
Fig. 2. Cibarial armatures and hard palates of male Sergentomyia sp. from Botswana. A+B.
7
Se. bedfordi “Maun” form. C. Se. salisburiensis. Scale bar 30 µm. (Nomarsky/DIC
8
microscopy).
us
cr
6
an
9
Fig. 3. An unrooted (neighbour-joining) tree of Botswana phlebotomine sandfly taxa, based
11
on comparison of sequences of mitochondrial gene fragments. A. COI. B. cytB. C. ND5.
12
Bootstrap values > 80% are shown in the tree on each node.
M
10
16 17 18 19 20 21
te
15
Ac ce p
14
d
13
22 23 24 25
19
Page 19 of 24
ce Ac
Page 20 of 24
M an ed ce pt Ac Page 21 of 24
u an M ed pt Ac ce Page 22 of 24
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ed
pt
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26 27 Highlights
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• For the first time, Phlebotomus rodhaini, a putative vector of leishmaniasis, is reported from Botswana • I total, five phlebotomine sandfly species are currently known from this country • The study provides baseline data for molecular typing of southern African sandflies, including the first ND5 mtDNA sequences for Afrotropical species
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S0001-706X(17)30061-X http://dx.doi.org/doi:10.1016/j.actatropica.2017.03.009 ACTROP 4235
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
17-1-2017 9-3-2017 10-3-2017
Please cite this article as: Krddotuger, A.,Phlebotomine sandflies of Botswana: A taxonomic review and a faunistic update with the first record of genus Phlebotomus, Acta Tropica (2017), http://dx.doi.org/10.1016/j.actatropica.2017.03.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Phlebotomine sandflies of Botswana: A taxonomic review and a faunistic
2
update with the first record of genus Phlebotomus
3 Andreas Krüger
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20359 Hamburg, Germany, and Okavango Research Institute, University of Botswana, P. Bag
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285, Maun, Botswana
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E-Mail: [email protected]
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Department of Tropical Medicine, Bundeswehr Hospital Hamburg, Bernhard-Nocht-Str. 74,
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RUNNING TITLE
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Updated sandfly records in Botswana
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ABSTRACT
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The first records of phlebotomine sandflies from Botswana have been published only
17
recently, comprising of four species of genus Sergentomyia. This update presents the first
18
record of genus Phlebotomus, namely Ph. (Anaphlebotomus) rodhaini Parrot, which is also
19
the first detection of a putative vector of leishmaniasis in Botswana. In addition, records of
20
the Sergentomyia “bedfordi (Newstead) group” are reviewed, and the molecular taxonomy of
21
all taxa known from Botswana is analysed based on three mitochondrial gene fragments
22
(mtDNA). The presence of Se. congolensis (Bequaert and Walrveus) and Se. salisburiensis
23
(Abonnenc) is confirmed, whereas the previously mentioned Se. caliginosa Davidson and
24
unassigned specimens of the “bedfordi group” are proposed to belong to the tentatively
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Present address 1
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named Se. bedfordi “Maun” form. The mtDNA analyses confirmed the species delimitations.
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For the first time, portions of the ND5 gene were used for the purpose of sandfly molecular
27
taxonomy. This gene revealed a high inter-specific variability and may thus be applied as an
28
alternative molecular marker for future studies.
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KEY WORDS
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Afrotropical, Phlebotominae, taxonomy, cytB, COI, ND5
1. Introduction
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Sandflies (Psychodidae: Phlebotominae) are haematophagous nematoceran Diptera that are, in
36
contrast to mosquitoes, blackflies and biting midges, independent from aquatic environments,
37
as their offspring develops strictly terrestrial, e.g. in animal burrows and shelters, rock cracks,
38
or caves. In many regions of the world they are obligate vectors of kinetoplastid parasites of
39
the genus Leishmania, the causative agents of leishmaniasis infection. Major human disease
40
foci are found across Africa (WHO, 2010), with the exception of southern Africa, where there
41
are only small and scattered foci known in Namibia (Noden and van der Colf 2013) and
42
Zambia (Alvar et al. 2012).
43
The knowledge on the Afrotropical sandfly fauna increased between 1951 and 1993 from 58
44
to 147 species and subspecies (Kirk and Lewis, 1951; Seccombe et al., 1993). The
45
zoogeographical southern African sub-region with the Rhodesian Highland, the Southeast
46
Veldt and the Southwest Arid districts (Kirk and Lewis, 1951), covering Namibia, Botswana,
47
Zimbabwe, South Africa, Lesotho, Swaziland, as well as parts of Angola, Zambia and
48
Mozambique harbours about 51 species, 32 of which are endemic to southern Africa
49
(Seccombe et al., 1993). Only recently the first records of phlebotomines from Botswana were
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reported (Krüger, 2015, 2016), namely Sergentomyia (Grassomyia) inermis (Theodor, 1938),
51
Se. (Ser.) congolensis (Bequaert and Walravens, 1930), Se. (Ser.) caliginosa Davidson, 1987
52
and Se. (Ser.) salisburiensis (Abonnenc, 1967), the latter three of which belong to the Se.
53
“bedfordi (Newstead, 1914) group”. If based on small sample sizes, misinterpretations of
54
morphological characters in these small-bodied and closely related species can easily happen
55
due to damaged body parts or suboptimal preparation of the microscopic slides containing the
56
structures of interest such as cibarial armature, pharynx and genitalia. Analyses of further
57
samples, and comparison with the initial specimens revealed the presence of two additional
58
taxa one of which replaces the erroneously recorded Se. caliginosa.
59
Taking advantage of the availability of specimens of closely related species from South
60
Africa, Namibia and Cameroon, data on the molecular taxonomy of Botswana sandflies were
61
also retrieved by conducting DNA sequence comparisons of the mitochondrial genes for
62
cytochrome b (cytB) and cytochrome c oxidase I (COI). The latter is the most commonly used
63
region for DNA barcoding (Hebert et al., 2003) and has previously been analysed in Indian
64
Sergentomyia (Pradeep Kumar et al., 2012), whereby cytB analyses have already been
65
conducted on sandflies from Afghanistan and elsewhere (Krüger et al., 2011). In addition, the
66
5’ portion of the mitochondrial gene for the NADH dehydrogenase subunit 5 (ND5) was
67
analysed for the first time in order to evaluate its suitability for phlebotomine taxonomy (Ye
68
et al., 2015).
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2. Material and methods
71 72
Details on the study area, sampling data, trapping technique, morphological sample
73
preparation and species identification of 28 specimens have been published recently (Krüger,
74
2015). In brief, the study area was in the vicinity of Maun, northern Botswana, the centre of
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Page 3 of 24
Ngamiland district. The region is very flat at an elevation of about 940 m above sea level, and
76
characterised by the unique combination of an inland river delta, surrounded by savannah and
77
semi-desert. The sampling device was a Standard CDC light-trap (Model 512;
78
John W. Hock, Gainesville, FL), deployed 50 cm beside a termite hill at a fan height of 50
79
cm, about 10-200 m off Thamalakane river. One collection site was in Sexaxa ward, in the
80
premises of the Okavango Research Institute (ORI), the second site in a private property in
81
Disaning ward. The trap was run from 5pm to 8.30am during seven trap nights.
82
An additional nine specimens (5 males, 4 females), whose analysis is reported here, have the
83
same details and were processed accordingly (Table 1).
84
Each mounted specimen was documented photographically and identified using the
85
morphological keys provided by Abonnenc (1972) and Davidson (1990), the latter from
86
which the taxonomy and terminology used here for the Sergentomyia “bedfordi group” was
87
adopted.
88
For genomic DNA extraction, ethanol-preserved, unmounted body parts (thorax and proximal
89
parts of the abdomen, legs, wings) of each sandfly of interest were dried for 3 hrs at 37°C, and
90
subsequently processed with the DNeasy blood & tissue kit (Qiagen, Hilden, Germany),
91
following Qiagen’s supplementary protocol for purification of total DNA from insects, using
92
disposable microtube pestles for homogenization. For comparison, further specimens of
93
closely related species from Afghanistan (Kondoz town, collected 2009; Krüger et al., 2011),
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Cameroon (Mokolo town, collected 2012, unpublished) and South Africa (KwaZulu Natal,
95
collected 2015 by A. Latif, unpublished) were processed, i.e. Se. distincta (Theodor, 1933),
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Se. schwetzi (Adler, Theodor & Parrot, 1929), Se. (Gra.) dreyfussi ssp. turkestanica Theodor
97
& Mesghali, 1964, Se. (Gra.) ghesquierei (Parrot, 1929), and Se. (Gra.) squamipleuris
98
(Newstead, 1912).
99
All PCRs were performed in a Biometra TProfessional Basis thermocycler (Biometra,
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Göttingen, Germany). The protocols for cytB and COI PCRs were as described previously
101
(Ready et al., 1997, Krüger et al., 2011, Pradeep Kumar et al., 2012), using GoTaq G2 Hot
102
Start and GoTaq G2 Flexi polymerase, respectively (Promega, Mannheim, Germany). The
103
only modification was for cytB reactions a 40 sec annealing during the first five cycles
104
(instead of only 30 sec; Ready et al., 1997). For ND5 amplification, an approximately 510
105
base pair long fragment at the 5’ end of the ND5 coding region was chosen, as this was shown
106
to be the most variable region of the entire sandfly mitochondrial genome (Ye et al., 2015).
107
The following primers were used: forward N5-J-6579 (modified from Simon et al., 1994)
108
TTCWCTYCAWCCTTGATC; reverse N5-6500 (modified from Birungi and Munstermann,
109
2002) GCBGGYTTTTATTCWAAGGA. PCR amplifications were performed in 50 µl
110
volumes containing 1 µl of DNA template, 9 µl Go Taq Flexi buffer (Promega, Mannheim,
111
Germany), 3 µl 25 mM MgCl2, 0.2 pmol/µl of each primer, 0.3 µl 10 mM dNTP mix, and 1.5
112
U of Go Taq Hot Start polymerase. The thermal cycler conditions were the same as for cytB
113
PCR (see above).
114
All reactions were run at 2.2% FlashGel cassette agarose gels (Lonza, Rockland, USA).
115
Positive reactions were purified with the NucleoSpin Extract II kit (Macherey-Nagel, Düren,
116
Germany) and commercially sequenced by direct forward and reverse sequencing (Seqlab,
117
Göttingen, Germany). The respective sequences were deposited in Genbank under accession
118
numbers KY451779-451798 (COI), KY451799-451811 (ND5), KY451812-451833 (cytB).
119
The cytB (422 base pairs) and ND5 (491 bp) sequences were individually run against the
120
BLAST tool of the publicly available NCBI Genbank in order to search for highly similar
121
sequences. Further, they were aligned using Clustal Omega
122
(http://www.ebi.ac.uk/Tools/msa/clustalo/) and analysed in MEGA7 (Kumar et al., 2016).
123
Neighbour-Joining (NJ) analyses were computed using the Kimura 2-Parameter method.
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Bootstrap values were calculated to test the robustness of the resulting taxon ID trees and
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were obtained by conducting 1000 replicates.
126
For COI barcoding, the obtained sequences (533 bp) were initially run against two databases,
127
i) by using the BLAST tool, and ii) the specimen identification tool of BOLD4 (Barcode of
128
Life Data systems v4 (http://v4.boldsystems.org/index.php). In order to visualize the
129
barcoding results, COI sequences were aligned and analysed as described above, in this case
130
together with Genbank entry JX105042 for Ph. rodhaini Parrot, 1930 and BOLD reference
131
sequence BNITM095-12 for Se. (Gra.) dreyfussi ssp. turkestanica from Afghanistan (detailed
132
specimen records and sequence information including traces files can be found within the
133
BOLD Working Group 1.4 Initiative “Human Pathogens and Zoonoses”: “Ticks and
134
Sandflies at BNITM Hamburg 2012”).
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139
In addition to the 28 previously investigated specimens, another nine were morphologically
140
identified to species level. The new specimens belonged to five species, three of which had
141
been reported previously (Table 1): Se. (Gra.) inermis (2 additional specimens), Se. (Ser.)
142
congolensis (1 more specimen) and Se. (Ser.) salisburiensis (2 additional specimens). One
143
specimen that was previously assigned to Se. (Ser.) caliginosa of the “bedfordi group”, six
144
specimens that were previously treated as “bedfordi group” sensu lato, and three new
145
specimens, turned out to belong to a new taxon tentatively named Se. bedfordi “Maun” form.
146
A single female fly was unequivocally identified as Phlebotomus (Anaphlebotomus) rodhaini.
147
Three of the recorded species belong to the Se. “bedfordi group” and are rather delicate to
148
distinguish. Updated morphometric measurements are summarized in Table 2. For
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comparison, data for Se. distincta, as well as published data for other relevant species (see
150
below) are listed, i.e. for Se. caliginosa, Se. formica Davidson, 1987 and Se. bedfordi s.str.
151
The decision for (re-)assigning 10 specimens to the new “Maun” form of Se. bedfordi was
152
based on the observation that both males and females showed character combinations close to
153
or intermediate between Se. caliginosa and Se. formica.
154
In females of the “Maun” form, the shape of the cibarial pigment plate resembles that of Se.
155
caliginosa and, to a lesser degree, of Se. bedfordi s.str., being oblong with a variable median
156
projection (Fig. 1). In addition, the posterior margins are strikingly ragged. However,
157
intermediate or Formica-like characters are as follows: cibarial armature with 41-50 closely
158
packed, long fine, monomorphic teeth [according to Davidson (1990) 45-65, versus 35-42 for
159
Se. caliginosa; see Table 2]; median posterior margin of hard palate with tuberculate
160
denticles, though small (smooth in Se. caliginosa); apical margin of hypopharynx with deep
161
indentations (weakly undulating in Se. caliginosa).
162
Males of the “Maun” form (n=3) of Se. bedfordi exhibited the following characters: cibarial
163
armature with 16-23 small, irregular teeth (16-24 in Se. caliginosa, versus 30-42 in Se.
164
formica); posterior margin of hard palate (Fig. 2A, B) with one to three rows of small
165
tuberculate denticles (large tooth-like eruptions in Se. formica, a cibarial anterior row of
166
spiculate denticles in Se. caliginosa); pigment plate absent (present in Se. formica);
167
Morphometrics (n=2): length of A3 125-134 µm; Ascoid-3 21-28 µm; Ascoid-4 21-23 µm.
168
Figure 2C shows the cibarial armature of a male Se. salisburiensis for comparison, with a
169
rather concave posterior margin of the hard palate bearing large tooth-like eruptions.
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170 171
3.2. Molecular taxonomy
172
All three mitochondrial DNA analyses unequivocally confirmed the morphological species
173
delimitations (Fig. 3), with high bootstrap support for the species clusters highlighting low
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intra-specific variability. Furthermore, the NJ genealogies are to a large extent in agreement.
175
In particular the Botswana members of the “bedfordi group”, i.e. the “Maun” form of Se.
176
bedfordi, Se. congolensis and Se. salisburiensis form a cluster together with Se. distincta from
177
Cameroon. Another cluster consists of Se. inermis from Botswana and the related species of
178
the subgenus Grassomyia, i.e. Se. dreyfussi turkestanica, Se. ghesquierei and Se.
179
squamipleuris, respectively. Phlebotomus rodhaini is consistently divergent from the other
180
taxa. In the COI analysis (Fig. 3A) however, Se. schwetzi from Cameroon is placed within the
181
“bedfordi group” cluster, which could be due to insufficient taxon coverage with regard to the
182
“schwetzi group” of related species (Davidson, 1990). Regarding the newly introduced ND5
183
analysis (Fig. 3C) it can be resumed that this gene region is highly variable between species,
184
resulting in slightly higher inter-specific genetic distances than in the COI and cytB regions,
185
as deduced from the branch length scale.
186
High similarity BLAST searches (megablast) of cytB sequences revealed high identity scores
187
for the reference specimens Se. dreyfussi turkestanica (100% with Se. dreyfussi turkestanica)
188
and Se. schwetzi (98% with Se. schwetzi), as well as for Ph. rodhaini (98% with Ph. rodhaini
189
from CAR). All other taxa had scores of ≦94%, indicating the absence of con-specific
190
Genbank entries. Megablast searches of COI sequences yielded a 99% identity score for Se.
191
schwetzi, whereas for Ph. rodhaini it was only 94% with another Ph. rodhaini, which
192
contained numerous undetermined base positions (“N”). For all remaining taxa the identity
193
scores only reached less than 93%, again indicating the absence of con-specific Genbank
194
entries. The ND5 sequences showed only up to 80% identity on blastn searches, whereby
195
Genbank at present only contained three sequences of three species of phlebotomine
196
sandflies.
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197 198
4. Discussion
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199 The most relevant finding of this study is the first record for Botswana of a putative vector of
201
zoonotic leishmaniasis, which is Ph. rodhaini. Elnaiem et al. (2011) incriminated this sandfly
202
species as a possible vector of Leishmania donovani in Sudan. Phlebotomus rodhaini is
203
widespread in sub-Saharan Africa including Namibia and South Africa, but was so far not
204
recorded in Botswana, Zambia, and Mocambique (Davidson, 1981, Niang et al., 2011). The
205
Botswana specimen, a gravid female, was collected at light next to a termite hill in the gallery
206
forest about 10 m off Thamalakane river in Maun. These conditions resemble those described
207
by Davidson (1981) for sampling sites in Namibia (Mashare, Kavango region) and South
208
Africa.
209
Concerning the Se. “bedfordi group” the species delimitation turned out to be challenging.
210
Abonnenc (1972) tried to shed light into the confusing taxonomy of the group then
211
comprising 12 entities. Davidson (1990) subsequently again revised the group, now
212
comprising 20 species. As an example of the persistent problems he added the following
213
comment to the female holotype of Se. bedfordi s.str.: “This specimen appears to be unique
214
and may be aberrant.” Contrary to Davidson (1990), Seccombe et al. (1993) retained certain
215
species as invalid under Se. bedfordi s.str., e.g. Se. congolensis and Se. distincta.
216
In the light of considerable morphological overlaps between the taxa (see Table 2), and the
217
modern approach of integrated morphological and molecular taxonomy it seems mandatory to
218
revise the “bedfordi group” again through a comparison of DNA sequences of typical
219
specimens when these become available. At least, typical Se. caliginosa and Se. formica
220
should be examined, as the newly designated “Maun” form could not be assigned to either of
221
the two on a morphologically basis, but which at least exhibited a clear molecular distance
222
from other members of the “bedfordi group”. Only then it can be proven whether the “Maun”
223
form constitutes a new species, or only a variant or part of a hybrid population of the before
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mentioned species. This could be supplemented by analysing at least one non-mitochondrial
225
marker (Depaquit, 2014).
226
Concerning the presence of species of the subgenus Grassomyia in South Africa there is a
227
slight confusion in Abonnenc (1972). The main text listed Se. inermis for this country, but the
228
respective distribution map only showed Se. squamipleuris in South Africa. The present study
229
revealed the latter to be true, but according to Abonnenc (1967) it seams reasonable to assume
230
both species to be present, with Botswana adding a second southern African record of Se.
231
inermis.
232
There is no obvious explanation why Botswana’s invertebrate fauna has received so little
233
attention, placing the country as a “white spot” on many faunal maps (Krüger and Deckert,
234
2016). By 2016, the only inventories publicly available were on butterflies, dragonflies,
235
grasshoppers and true bugs, leaving most insect taxa seemingly as absent in the country,
236
including Isoptera (termites) or sandflies (Republic of Botswana, 2015; Krüger, 2015; Krüger
237
and Deckert, 2016).
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Acknowledgement
240
Annett Michel is gratefully acknowledged for excellent technical assistance. Professor
241
Abdalla Latif is thanked for giving specimens. Parts of this study with regard to Cameroon
242
specimens were funded by the Volkswagen foundation.
243 244
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239
References
245 246
Abonnenc, E., 1967. Révision des phlébotomes de l’Afrique au sud du Zambese, cle de
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determination et description de: P. salisburiensis n. sp., P. zumpti n sp., et P. haeselbarthi n
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Abonnenc, E., 1972. Les phlébotomes de la région Èthiopienne (Diptera, Psychodidae). Mém.
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O. R. S. T. O. M. 55, 289 pp.
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252 Adler, S., Theodor, O., Parrot, L., 1929. Phlébotomes du Congo Belge. Rev. Zool. Bot. afr.
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Depaquit, J., 2014. Molecular systematics applied to Phlebotomine sandflies: review and
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perspectives. Infect. Genet. Evol. 28, 744-756. doi:10.1016/j.meegid.2014.10.027.
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transmission of Leishmania donovani. Parasit. Vectors. 21(4), 238. doi:10.1186/1756-3305-4-
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Hebert, P.D.N., Ratnasingham, S., deWaard, J.R., 2003. Barcoding animal life: cytochrome c
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oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B Biol. Sci.
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Krüger, A., 2016. First Phlebotominae in Botswana. In: Depaquit, J., Pesson, B., Augot, D.,
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Krüger, A., Strüven, L., Post, R.J., Faulde, M., 2011. The sandflies (Diptera: Psychodidae,
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Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular Evolutionary Genetics
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Parrot, L., 1929. Sur un autre phlébotome nouveau du Congo Belge Phlebotomus ghesquierei
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Weighting, and Phylogenetic Utility of Mitochondrial Gene Sequences and a Compilation of
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Conserved Polymerase Chain Reaction Primers. Ann. Entomol. Soc. Am. 87(6), 651-701. doi:
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https://doi.org/10.1093/aesa/87.6.651.
343
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344
Theodor, O., 1933. Some African sandflies. Bull. Ent. Res. 24, 537-547.
345 346
Theodor, O., 1938. On African sandflies III. Bull. Ent. Res. 29, 165-173.
348
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347 Theodor, O., Mesghali, A., 1964. On the Phlebotominae of Iran. J. Med. Ent. 1, 285-300.
350
cr
349 WHO, 2010. http://www.who.int/leishmaniasis/leishmaniasis_maps/en/.
us
351
Ye F., Liu T, King, S.D., You, P.. 2015. Mitochondrial genomes of two phlebotomine sand
353
flies, Phlebotomus chinensis and Phlebotomus papatasi (Diptera: Nematocera), the first
354
representatives from the family Psychodidae. Parasit. Vectors. 8, 472. doi: 10.1186/s13071-
355
015-1081-1.
M
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i cr us
Date
Maun, Disaning, Termite hill
15.-16. xii 2014
Maun, ORI, Termite hill
26.-27. xi 2014
1f
Congolensis
"Maun" form
2f (1f)
1f
1.-2. xii 2014
2f
3.-4. xii 2014
2f
1f
9.-10. xii 2014
5f, 1m
3f
13.-14.i 2015
1f, 1m
ce
Salisburiensis
1f
2f, 2m
pt
S19°53'59" E23°31'43"
10.-11. i 2015
Inermis
1f, 1m
ed
S19°57'58" E23°27'43"
Rodhaini
M
Location
an
Table 1. Summary of sampling locations, dates and numbers of collected sandfly species. Bold numbers: sequenced specimens.
1f, 1m 1m
1m
1f 2f, 1m
1f
1f
1f
12f, 4m
6f, 1m
2f, 1m
7f, 3m
TOTAL
1
16
7
3
10
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Sub-TOTALS
16 Page 16 of 24
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an
Table 2. Morphometric measurements of Sergentomyia “bedfordi group” females from Botswana, with counting of the cibarial teeth, lengths of antennal segments A3, A4 and A5. For comparison, further members of the group, which are either mentioned in the text or used for analyses,
M
are listed. SE: standard error; n: number of specimens measured. All lengths measures are given in micrometer.
Species:
ed
Botswana
A4
bedfordi s.str.
bedfordi “Maun”
distincta
distincta
formica
caliginosa
holotype
Range (n)
30-38 (5)
29-37 (2)
41-50 (5)
17-24 (17)
20-30 (30*)
45-65 (30*)
35-42 (10*)
20
Mean (SE)
35 (1.72)
--
43.8 (1.85)
21.8 (0.54)
--
--
--
--
Range (n)
120-136 (5)
127
105-122 (4)
105-135 (5)
100-140 (30*)
95-125 (30*)
90-120 (10*)
165
Mean (SE)
128.2 (3.35)
--
112.5 (3.57)
116 (5)
--
--
--
--
Range (n)
70-77 (5)
79
65-70 (4)
65-80 (5)
59-79 (30*)
56-72 (30*)
55-70 (10*)
87
Ac
A3
ce
Character Cibarial teeth
salisburiensis
According to Davidson (1990):
pt
congolensis
Cameroon
17 Page 17 of 24
i cr 70-80 (5)
90
Mean (SE)
75.2 (1.59)
--
72 (2)
--
--
--
--
68-70 (3)
70-85 (5)
--
--
--
--
69.3 (0.67)
76 (4)
--
--
--
--
Ac
ce
pt
* includes holo- or lectotype.
us
Range (n)
67 (1.22)
an
--
M
73.2 (1.12)
ed
A5
Mean (SE)
18 Page 18 of 24
Fig. 1. Cibaria of female Sergentomyia bedfordi “Maun” form from Botswana. The dark
2
structures in the centre represent the pigment plates. Right panel: same specimen at different
3
focus levels, above for pigment plate, below for cibarial armature with 41 teeth.
4
(Nomarsky/DIC microscopy).
ip t
1
5
Fig. 2. Cibarial armatures and hard palates of male Sergentomyia sp. from Botswana. A+B.
7
Se. bedfordi “Maun” form. C. Se. salisburiensis. Scale bar 30 µm. (Nomarsky/DIC
8
microscopy).
us
cr
6
an
9
Fig. 3. An unrooted (neighbour-joining) tree of Botswana phlebotomine sandfly taxa, based
11
on comparison of sequences of mitochondrial gene fragments. A. COI. B. cytB. C. ND5.
12
Bootstrap values > 80% are shown in the tree on each node.
M
10
16 17 18 19 20 21
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15
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14
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22 23 24 25
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ce Ac
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M an ed ce pt Ac Page 21 of 24
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ed
pt
ce
Ac M
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26 27 Highlights
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• For the first time, Phlebotomus rodhaini, a putative vector of leishmaniasis, is reported from Botswana • I total, five phlebotomine sandfly species are currently known from this country • The study provides baseline data for molecular typing of southern African sandflies, including the first ND5 mtDNA sequences for Afrotropical species
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28 29 30 31 32 33 34 35 36 37
20
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