Morphological, molecular identification and distribution of trypanosome-

transmitting dipterans from cattle settlements in southwest Nigeria

First author: Paul Olalekan ODENIRAN1,2

Second author: Ewan Thomas MACLEOD2

Third author: Isaiah Oluwafemi ADEMOLA1

Fourth author: John Asekhaen OHIOLEI3

Fifth author: Ayodele Oluwakemi MAJEKODUNMI2,4

Sixth author: Susan Christiana WELBURN2,4

Institution addresses:

(1) Department of Veterinary Parasitology and Entomology, Faculty of Veterinary

Medicine. University of Ibadan, Nigeria.

(2) Infection Medicine, Biomedical Sciences, University of Edinburgh, United Kingdom.

EH8 9JZ.

(3) State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary

Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese

Academy of Agricultural Sciences, Lanzhou 730046, People’s Republic of China.

(4) Zhejiang University – University of Edinburgh Joint Institute, Zhejiang

University, International Campus, 718 East Haizhou Road, Haining, 314400, China

Running Title: Identification of vectors of bovine trypanosomiasis in southwest Nigeria.

Corresponding author: Paul Olalekan ODENIRAN

Email address: drpaulekode@gmail.com

Phone number: +2347068467479

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Abstract

Introduction Glossina spp. (Glossinidae), Tabanus spp. (Tabanidae), Ancala spp.

(Tabanidae), Atylotus spp. (Tabanidae) and Stomoxys spp. (Muscidae) are important

transmitting vectors of African animal trypanosomosis in sub-Saharan Africa. There is

paucity of information on the distribution and identification of these flies in cattle settlements

in southwest Nigeria.

Methods The distribution patterns, genetic variations and diversities of dipteran flies in

southwest Nigeria were described and identified using morphological and molecular analysis

of the 28S rDNA gene.

Results Of the 13,895 flies examined morphologically between April 2016 and March 2017,

tabanids were identified [Tabanus (0.34%), Ancala (0.03%), Atylotus (0.01%), Haematopota

(0.014%) and Chrysops (0.11%)]. Two stomoxyine species were identified; Stomoxys niger

niger Macquart (45.30%) and Stomoxys calcitrans Linnaeus (17.29%) and two Glossina spp.

namely; Glossina p. gambiense Vanderplank, 1911 (0.46%) and Glossina tachinoides

Westwood (0.51%) were identified. The identities were further confirmed in a BLAST search

using their nucleotide sequences. The median-joining network of the 28S rDNA gene

sequences indicated that fly species examined were genetically distinct. The apparent density

of all the trapped flies was highest at a mean temperature of 26 – 28°C, humidity >80% and

rainfall of 150 – 220 mm/month. The distribution of flies was observed to increase as

vegetation increased in density and decreased in areas with relatively high human population

density (>100/km2).

Conclusions The population indices of the 28S rDNA gene of the flies suggest that analysis

of nuclear DNA fragments may provide more information on the molecular ecology of these

flies. Characterising fly species and assessing their impact is essential in distribution and

monitoring AAT spread.

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Keywords Tsetse flies, Horse fly, Stable fly, Morphology, Phylogenetics, Southwest Nigeria

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Introduction

Human African trypanosomiasis (HAT), also known as sleeping sickness and animal African

trypanosomiasis (AAT) also known as nagana occurs throughout sub-Saharan Africa. Over

60 million people and 48 million cattle are considered at risk of infection across an area of 10

million km2. Limited surveillance leads to under-detection in both humans [1] and livestock

[2] and as such these diseases are considered to cause an estimated 5,000 human and 3

million livestock deaths per annum [3, 4].

Tsetse flies, Glossina spp. (Diptera: Glossinidae) are biological vectors of trypanosomes that

cause HAT and AAT [5, 6]. Glossina is classified into three species groups; Palpalis

(Nemorhina), Morsitans (Glossina) and Fusca (Austenina) [7]. Nigeria is endemic for the

chronic form of HAT (Trypanosoma brucei gambiense) and AAT (T. b. brucei,

Trypanosoma vivax and T. congolense) and is home to 11 known species of tsetse [7-11].

Diptera of the families Tabanidae and Muscidae [12], have been reported as mechanical

vectors of AAT [13, 14]. The family Tabanidae comprises four subfamilies (Chrysopinae,

Pangoniinae, Scepsidinae and Tabaninae), 144 genera and 4400 species [15, 16]. Members of

the Chrysopinae and Tabaninae transmit several infections of economic importance including

Equine infectious anaemia, loiasis, anthrax, tularaemia and trypanosomiasis [17-19].

However, tabanids remain one of the least studied groups of Diptera and there are few

taxonomic studies of Tabanus in Nigeria [20, 21].

There are 18 species of Stomoxys spp. (Diptera: Muscidae), commonly known as stable flies,

of which twelve are exclusive to Africa [22, 23]. Stomoxyine flies are similar in size and

colour pattern to house flies but have a pointed proboscis for piercing and sucking blood [24].

Both sexes are blood-sucking and feed on livestock, wildlife and humans [25]. They cause

irritation and significantly reduce weight gain and milk production in livestock [26].

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Stomoxys calcitrans, the most common species, has been shown to mechanically transmit

Trypanosoma evansi, Trypanosoma vivax and Trypanosoma brucei in laboratory studies [27].

They are also able to transmit many other pathogens, including Besnoitia besnoiti,

Anaplasma marginale, Bacillus anthracis, Coxiella burnetti and Habronema microstoma

[28]. Few studies have been reported in Nigeria [29-32].

A complete understanding of the patterns and factors contributing to Glossina, Tabanids and

Stomoxys distribution is essential for effective control of trypanosome and other arthropod-

borne infections but requires accurate vector identification. Vector identification is often

based on examination of genital morphology and wing and body colour [19] and the

hypervariable nature of many of these physical characteristics, coupled with body colour

changes during sample storage and a lack of defined morphological keys compromises

identification [33-35]. Stomoxys species have been misidentified in a number of studies [19,

36]. Hence, there is a need for molecular determination of flies due to the significant

limitations of morphological characteristics.

The first aim of this study was to characterise trapped Glossina, Tabanids and Stomoxys from

southwest Nigeria applying both morphological and molecular techniques for identification.

The second aim was to understand the ecological influence on the abundance and distribution

of these flies.

Materials and methods

Study area

The study area is ~ 78,000km2, between latitudes 6°64'04.66"N - 7°67'77.14"N and

longitudes 2°75'11.18"E - 5°20'46.13"E within the transition zone from derived savannah to

lowland rainforest vegetation (Fig. 1). Between April 2016 and March 2017, fifteen livestock

establishments in ten towns were visited. The livestock settlements were selected randomly

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across the southwest Nigeria from the cattle settlement lists. All the study sites were visited in

the wet (April - October) and dry (November - March) season.

Livestock settlements in Igangan, Eruwa, Igboora and Akingbite are owned by the Fulani

pastoralists. However, in these areas they practice mainly pastoralism and, in few occasions,

some engage in transhumance in the wet season. In Akingbite, two settlements were visited.

Here, cultivation is intense, dominated by local farmlands. Riverine vegetation occurs only in

narrow strips often cleared at many points. Fulani settlements in Eruwa occur in patches,

cultivation is mild while the two study sites were surrounded with secondary forest. Igangan

livestock settlement is characterised with large expanse of land that encamps several discrete

Fulani pastoralists. Here, three study sites were located and the patchy vegetation is a dry

tropical rainforest, with cattle population in excess of 10,000. Igboora farm is about 70 km

away from Eruwa, with slightly dense vegetation. The cattle herd was about 100 in capacity

and owner is often involved in transhumance during the wet season with take-off in February.

The study sites in Federal University of Agriculture, Abeokuta, Ogun State (FUNAAB) and

University of Ibadan, Oyo State (UI) are institutional livestock establishments in which cattle

are kept under semi-intensive system. The areas are in wet tropical rainforest zone with

scanty secondary forest. No wildlife was seen and cattle population was around 200 in each

site. Ponpoola, Adebayo and Idiroko study sites are owned by wealthy community elite who

employed the services of the Fulbe to engage the animals in pastoralism, however, there is a

settlement were the animals are kept and treated. Idiroko and Adebayo have primary forest

around but the farms are fenced. Vegetation is dense and cattle population is approximately

1000 and 100 in Idiroko and Adebayo, respectively. In Ponpoola, however, two study sites

were involved and cattle population was 150 and 113, respectively. Sango, Akure study site

is a cattle market establishment were hundreds of cattle from northern part of the country are

brought for sale. Traps were set in strategic areas of the market.

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Fly capture and sample processing

Flies were trapped using non-baited Nzi traps [37] designed locally using Sunbrella® Pacific

Blue, Sunbrella® Black, white polyester mosquito mesh. The basic Nzi trap design was

modified by attaching an improvised plastic collecting jar to the top of the trap. Four traps

were set per cattle settlement study site, 500 m equidistant from each other and flies were

collected every 12 hours (7 am and 7 pm) to avoid desiccation. Traps were set for five

consecutive days each season (wet and dry) for all the study sites. Hence a total of 120 traps

were set in the cattle settlements. Trapped flies were air dried and then preserved in 95%

ethanol.

Taxonomical determination of species

Preserved flies were examined using a graduated reticule embedded stereomicroscope

(Euromex®, UK) and photographed with a Nikon A900 digital camera. Specimens were

morphologically identified to species level according to Systema Dipterorum. Particular

attention was paid to two important aspects; (i) alignment of ocular micrometre with

morphological landmarks, (ii) proper transcription and conversion of ocular units to metric

equivalents. Glossina spp. were characterised using identification keys including arista, shape

of superior and inferior claspers and abdominal markings [38, 39], while Stomoxys spp. were

identified using wing patterns, thoracic and abdominal markings, limb shape and colour,

frons, mouth-parts and genitalia [22]. Tabanus species were identified using several keys

from different authors [39-43]. Body description which involves shape, colour, sizes of

distinct parts of the body such as head, thorax, abdomen, limbs, mouth-parts and wings were

principal taxonomical characters.

Molecular identification of flies and DNA sequencing

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Flies were surface sterilised using 5% sodium hypochlorite and phosphate-buffered saline

(NaCl 8.0 g/L, KCl 0.2 g/L, Na2HPO4 1.15 g/L, KH2PO4 0.2 g/L; pH 7.3) (Sigma-Aldrich

Chemie GmbH, Munich, Germany). The whole fly was then homogenised using a pestle in a

1.5ml Eppendorf tube. DNA was then extracted using a Qiagen DNeasy blood and tissue kit

(Qiagen, Germany).

Molecular identification of the vectors (Glossina, Tabanids and Stomoxys) was based on

nuclear data sequences of internal transcribed spacer (ITS-2) rDNA using general insect

primers: ITSA - TGT-GAA-CTG-CAG-GAC-ACA-T; ITSB - TAT-GCT-TAA-ATT-CAG-

GGG-GT [44]. The oligonucleotide primers were constructed on an Applied Biosystems

(Forster City, CA) 394 DNA/RNA Synthesizer. PCR amplifications were carried out in 25μl

total volume of reaction mixture containing 5 μl of 5 × PCR buffer, 0.6 µl of 50 mM MgCl2,

16.6 µl of double distilled water, 0.2 µl of 25 mM dNTPs, 0.1 µl of 5U/µl units Taq DNA

polymerase, 0.75 μl of 10 pmol/μl each of primers and 1 μl of DNA template. Laboratory

Glossina palpalis DNA from Cameroon was used as positive control while distilled water

was used as negative control. PCR thermal cycling consisted of initial denaturation of 94°C

for 5 mins; annealing at 94°C for 1 min, 52°C for 1 min and 72°C for 2 mins at 30 cycles;

final extension at 72°C for 5 mins. Amplified PCR products were separated by 1.2% agarose

gel electrophoresis containing GelRed nucleic acid stain (Biotium Inc., USA) along with low

range exACTGene™ 100 bp molecular ladder (Fisher Scientific, UK).

Subsequently, PCR gel products were purified using the QIAquick PCR gel extraction kit

(Qiagen, Germany) and products were sequenced unidirectionally in GATC Biotech

(Germany) using Sanger sequencing. BLASTn searches were used to identify 28S rDNA

gene entries in the GenBank database with the closest nucleotide sequence.

Sequences were trimmed and aligned using ClustalW Multiple Alignment algorithm of

Unipro UGENE v1.29.0 software. Neutrality and diversity indices were estimated in DnaSP

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v.6 [45]. A network file was generated for each sequence dataset in DnaSP v6 [45],

considering gaps/missing sites and the median-joining network was constructed with

PopART [46], using the generated file. The best fit sequence substitution model for Glossina,

Tabanids and Stomoxys sequence datasets was HKY+G and HKY, respectively as determined

by jModelTest [47], based on the Bayesian information criterion (BIC).

Data analysis

Fly distribution across the study sites were examined using QGIS software (version 2.18).

The phylogenetic trees were then inferred with MrBayes v.3.1.2. Markov Chain Monte Carlo

(MCMC) sampling was used to assess the posterior distribution of parameters with a chain

length of 2,000,000 states, and 25% was discarded as ‘burn-in’. Parameters were logged

every 1000 states. The phylogenetic tree was displayed using TreeView v.1.6.6.

(http://taxonomy.zoology.gla.ac.uk/rod/treeview.html)

A total of twenty-seven Tabanus samples were sequenced from eight trapped species. Twenty

samples of Glossina spp., and twenty-four samples of Stomoxys spp., were sequenced from

the amplified DNA samples.

Apparent density was calculated to evaluate relative abundance as flies trapped per day [39].

Chi-square analysis was used to compare the abundance of flies using Graphpad Prism

(Graphpad Software, USA). Confidence intervals were calculated to determine 95%

confidence intervals with WINPEPI statistical package (UK).

Results

Morphological studies of transmitting vectors of trypanosomosis

Tabanus species (Diptera: Tabanidae)

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The Tabanus species identified morphologically were T. gratus (Loew, 1858), T. taeniola

(Palisot de Beauvois, 1807), T. subangustus (Ricardo, 1908), T. par (Walker, 1854), T.

biguttatus (Wiedemann, 1830), T. pertinens (Austen, 1912) and T. thoracinus (Palisot de

Beauvois, 1806).

Ancala species (Diptera: Tabanidae)

The only identified species was Ancala fasciatus (Fabricus)

Atylotus species (Diptera: Tabanidae)

Atylotus agrestis (Wiedemann) was identified

Stomoxys species (Diptera: Muscidae)

The two Stomoxys species observed in the study were Stomoxys calcitrans (Linnaeus, 1758)

and Stomoxys niger (Macquart, 1851).

Glossina species (Diptera: Muscidae)

Morphologically identified Glossina spp. from this study are Glossina palpalis spp. and

Glossina tachinoides (Westwood, 1850).

Phylogenetic analysis of transmitting vectors of trypanosomosis

The variation observed in the partial region of the nuclear 28S ITS gene and distinct clusters

formed were due to unique nucleotide sequence of individual species. Closely related species

were identified from common nodal points (Fig. 2). Sequenced Tabanus species results were

submitted to GenBank (MF448236, MF448237, MF448238, MF448239, MF448240,

MF448241).

Glossina species phylogenetic relatedness

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Of the 13 G. palpalis PCR amplicons that were sequenced, all aligned with G. palpalis

gambiense (Vanderplank, 1911) on the phylogenetic tree. Similarity matches of 98 and 99%

were observed during BLAST search. Similarly, the seven G. tachinoides sequences showed

high similarity matches (98-100%). Glossina p. gambiense were observed in two sub-clades

with pp values of 87% and 99%. Glossina tachinoides also displayed two subclades with pp

values of 100 and 50% respectively. Of the successfully amplified and sequenced samples,

the useable nucleotide sequences of Glossina spp. after correction and editing was 291 bp.

The Bayesian phylogeny constructed based on the sequences further confirmed the identity of

the flies as they clustered with other reference sequences from GenBank database and

exhibited significant distances from other Glossina species (Fig. 2). The median-joining

networks of Palpalis group of Glossina species from southwest Nigeria compared to other

species is shown in Fig. 3. It depicts the interspecific variation of the 28S rDNA revealing

several connections between haplotypes representing each species and the possible missing

mutational links or unsampled flies. In the network, each Glossina species cluster separately

with few mutation steps from each other except G. morsitans cluster that showed >16

mutation steps away from other species. Analysis of the 28S rDNA revealed one (parsimony-

informative) and three mutations (parsimony-informative) for G. palpalis and G. tachinoides,

respectively. No deletions or insertions were observed. Low haplotype (Hd) and nucleotide

(π) diversities were observed while Fu’s F and Tajima’s D were 0.240 and -0.27429,

respectively for G. palpalis and 2.920 and 1.81122, respectively for G. tachinoides (Table 1).

Tabanids phylogenetic relatedness

Only 9 out of 24 sequences retrieved with 97-99% similarity with database sequences were

used for the phylogenetic tree. There were limited sequence vouchers for Tabanus species in

the database for 28S rDNA. Molecularly characterised and submitted Tabanids include; TS6

(Tabanus gratus- MF448237), TS9 (Atylotus agrestis- MF448238), TS10 (Tabanus

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pertinens- MF448236), TS23 (Tabanus taeniola- MF448239), TS25 (Tabanus subangustus-

MF448240) and TS30 (Tabanus thoracinus- MF448241), assigned accession numbers from

GenBank (Fig. 4). The sequences of the 28 ITS2 gene of Tabanus species revealed two main

clusters or subclades with varied posterior probability (pp) values. The observed polymorphic

sites between species resulted in amino acid change. Within each cluster, similar species

showed high nodal support values of 98-100%. Other species like Atylotus agrestis, Ancala

fasciata, Tabanus biguttatus and T. par were not included in the phylogeny because of their

very short sequence length (Fig. 4).

Phylogenetic analysis for Stomoxys species

Morphologically identified S. niger sequences were recognised as Stomoxys niger niger, with

98 – 99% similarity (n = 13), while S. calcitrans sequences showed 96 – 99%

identity/similarity (n = 11). Only one sub-class clade was representative of S. calcitrans with

a pp value of 100%. Stomoxys niger niger showed two main sub-clades (Fig. 5).

Distribution and abundance of AAT vectors

A total of 13, 895 dipteran flies were captured in this study comprising 5,551 males and

8,344 females. The sex ratio of trapped flies indicates 60.1% (95%CI: 59.2 – 60.9%) female

and 39.9% (95%CI: 39.1 – 40.7%) male for total catches. Of these total flies, 64.7% (95%CI:

63.9 – 65.5%) were haematophagus flies of which 60.4% (95%CI: 59.4 – 61.4%) were

female. Trypanosome transmitting vectors (Glossina, Tabanids and Stomoxys) abundance in

relation to species distribution and sex have been reported (Table 2). Non-biting flies caught

during the survey were Musca domestica, Fannia spp., Chrysomyia spp. and Lucilia spp.

Musca domestica was most abundant (77.9%, 95%CI: 76.7 – 79.0%) of the non-biting flies

(Table 3).

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Catches were greatly reduced during dry periods with slightly higher temperatures. In areas,

with limited human population, tabanid catches were abundant unlike densely populated

human locations. However, Stomoxys spp. were less affected by human population, rather the

presence of cattle, especially areas where the paddock is filled with cattle dung. Tsetse were

trapped mostly during the wet season.

Glossina palpalis and G. tachinoides were morphologically identified in Adebayo, Oyo State

and Idiroko, Ogun State. Of 135 Glossina flies captured, 52.6% were identified as G.

tachinoides and 47.4% were G. palpalis. The apparent fly density for G. palpalis was 0.5

flies/trap/day during the wet season and 0.08 flies/trap/day in the dry season. The apparent

density of G. tachinoides was 0.6 flies/trap/day in the wet season and 0.02 flies/trap/day in

the dry season. The highest apparent density of 1.8 flies/trap/day was observed in May during

the wet season. Chi-square analysis of seasonal variation revealed that Glossina spp.

abundance was favoured in wet season (Χ 2 = 5.76, P = 0.016, OR = 8.8 (95% CI: 1.5-52.3))

compared to the dry season. The highest tsetse catches were recorded when rainfall was

between 150 – 220 mm/month and temperature < 30°C, while lowest when temperature was

> 30°C and rainfall below 60 mm/month. Low apparent densities of 0.0 – 0.3 flies/trap/day

were recorded in the dry season.

Flies were identified from three genera in the family Tabanidae: Tabanus (68.1%), Ancala

(5.8%), Atylotus (1.4%), Chrysops (21.7%) and Haematopota (2.9%). In total 47 Tabanus

spp. from seven taxa were captured (Table 3). T. taeniola was observed only during the wet

season and never captured when temperature was over 30°C and humidity less than 65%.

The Chrysops identified were C. distinctipennis Austen (Diptera: Tabanidae), C. longicornis

Macquart (Diptera: Tabanidae) and C. silacea Austen (Diptera: Tabanidae). The only

identified Haematopota was H. pertinens Austen (Diptera: Tabanidae). Tabanus spp. was

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also influenced by environmental factors. More Tabanids were captured during the wet

season (Χ 2 = 55.5, P < 0.0001, OR = 41.3 (95% CI: 13.6-126.1) compared to the dry season.

Tabanus taeniola Palisot de Beauvois (Diptera: Tabanidae) was the most abundant tabanid

captured in Akingbite, Eruwa, UI, Igangan and Igboora. T. subangustus Ricardo (Diptera:

Tabanidae) was the most widely distributed, trapped in Akingbite, Eruwa, UI, Igboora,

Idiroko and Sango in Akure. No tabanids were captured at FUNAAB, Abeokuta despite an

apparent density of > 50 fly per trap of Stomoxys species trapped in the location. Stomoxys

niger and Stomoxys calcitrans were observed in all of the areas sampled.

In total 8,697 Stomoxys spp. were trapped comprising Stomoxys niger (72.4%) and Stomoxys

calcitrans (27.6%). Chi-square analysis shows that there is significant increase (Χ 2 = 3485.2,

P < 0.0001, OR = 0.15 (95% CI: 0.14-0.16)) in the abundance of S. niger compared to S.

calcitrans. The relative abundance of Stomoxys spp. was at its greatest when the temperature

was 27 – 28°C, humidity was greater than 80% and rainfall was 100 – 250 mm. Seasonal

variation analysis showed that more Stomoxys spp. were captured in the wet season (Χ 2 =

16.7, P < 0.0001, OR = 0.75 (95% CI: 0.7-0.9) compared to the dry season. Both species also

favoured wet season compared to dry season, for instance analysis showed S. niger (Χ 2 =

5666.9, P < 0.0001, OR = 25.8 (95% CI: 23.5-28.3) and S. calcitrans (Χ 2 = 2644.1, P <

0.0001, OR = 45.5 (95% CI: 38.5-53.9)), respectively. Although, the collections in dry season

was 15.5% (95% CI: 14.7-16.3) of total Stomoxys population trapped.

On closer examination of all the haematophagus flies, 26.1% (95% 25.2-27.0) were fed.

Other non-biting flies collected which are not vectors of trypanosomiasis accounts for 35.3%

(95% CI: 34.5-36.1) out of overall 13,895 flies captured in the study. Hence, haematophagus

flies were significantly abundant (Χ 2 = 2394.9, P < 0.0001, OR = 0.30 (95% CI: 0.28-0.31))

compared to the non-biting flies. Stomoxys spp. account for 96.7% of total haematophagus

flies captured in this study.

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Discussion

In this study, identification of trypanosome-transmitting dipteran flies in southwest Nigeria

was addressed by morphological and molecular techniques. Correlation was observed

between both methods. Morphology considers the gross structure of organism or taxon and its

component parts, differentiating between species [48], while molecular characterisation has

become a standard tool for identification of dipteran flies due to its precision and accuracy [9,

19, 49]. The variable ITS2 rDNA primers have been used to phylogenetically characterise

Diptera: Calliphoridae [50] and Culicidae [51]. Most of the flies were trapped in the wet

months, with low catches observed in dry months as reported in previous studies [19, 30, 52].

The significant female bias sex ratio in the collection could be due to high haematophagus

flies in cattle settlements in which the female flies need blood for egg development.

In this study, the Glossina species were observed to be Glossina palpalis and Glossina

tachinoides morphologically. Although the two palpalis subspecies can be differentiated

through the ratio of the length to width of the dorsal plates, however; there can be overlap

[53]. Therefore, using morphometric analysis might not be a reliable tool to differentiate

them. Based on the location on phylogenetic tree and the BLAST search results, the species

trapped in this study were identified as G. p. gambiensis. Hence, the ITS-2 primer sequences

were used in the construction of the phylogenetic trees. Molecular identification therefore

showed an advantage over morphological method in classifying and identifying flies.

The low haplotype and nucleotide diversity depict a low genetic variation and diversity

among Glossina species population in Nigeria which has also been observed elsewhere based

on nuclear and mitochondrial DNA [61]. The neutrality indices for the G. palpalis population

as seen in the low negative and insignificant Tajima’s D and positive Fu’s F indicates an

excess of low-frequency polymorphism which suggests a recent population expansion or a

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recent recovery from a population bottleneck event as was recently reported in a Glossina

species population in Uganda [6]. The G. tachinoides population showed a positive but

insignificant Tajima’s D and Fu’s F suggesting the possibility occurrence of a population

bottleneck event in the course of their history. Although the sample size used in estimating

the population dynamics was small, other factors such as vector control programmes have

also been reported to influence Glossina population dynamics [6].

Palpalis group of Glossina observed from this study are known for their superior ability in

relation to changing conditions, particularly anthropogenic change, compared to Fusca or

Morsitans group [54]. The distribution of Glossina species were observed in patches of

forested riverine areas in Idiroko and Adebayo which was influenced by host availability,

flowing rivers and vegetation cover. The abundance of Glossina species in the wet season in

these target areas and consequent risk of trypanosomiasis transmission may be greatest at the

bioclimatic optimum for each species in relation to the site of capture [55, 56].

Thirteen Tabanid species belonging to five different genera were observed in this study

namely; Tabanus, Ancala, Atylotus, Chrysops and Haematopota. The majority (68.1%) were

Tabanus spp., known for mechanical transmission of trypanosomes [48]. The last study of

Tabanus species in southwest Nigeria was four decades ago [29]. Two species observed in

this study were not reported by Dipeolu [29]: T. par and T. gratus. Conversely, three species

reported by Dipeolu [29] were not observed in this study: T. socialis, T. neocopinus and T.

pluto. Four species, T. taeniola, T. biguttatus, T. thoracinus and T. subangustus were reported

from both studies. The changing climate and cattle management practices over the years

could be responsible for the differences in Tabanus species collection. The low overall

numbers of tabanids trapped could be because the collections were restricted to stationary

traps unlike previous work that combined stationary traps with hand nets.

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T. taeniola Palisot de Beauvois was the most abundant species observed in this study, which

is similar to previous reports [29, 42]. It is reported to be the most abundant in Africa, with

wide range distribution in Egypt, Sudan, Senegal to Delagoa Bay (Mozambique) and the

Transvaal (South Africa) [40]. This was reported during subsequent national survey of

Dipteran flies in Nigeria [29]. Tabanus subangustus Ricardo, the most widely distributed

species observed from this study was first to be reported in Nigeria [40]. Changes in

agricultural practices could have direct effect on the spread. A later report suggested its

abundance in both northcentral and southwest Nigeria [42]. Even though it was only found in

Ilorin four decades ago [29], it was not an established species in southwest Nigeria at that

time. The differences in distribution could be due to vegetational changes and human

activities. Ancala fasciata formerly reported as T. fasciatus Fabricus in Nigeria was found in

both seasons in this study in Adebayo area (northwest border between Oyo and Ogun state)

and University of Ibadan. The obvious deep green eyes without band, black ventral abdomen

and wing markings were distinctive. It can easily be confused with T. brucei Ricardo

(Diptera: Tabanidae), T. africanus Gray (Diptera: Tabanidae) and T. latipes Macquart

(Diptera: Tabanidae), due to the homogenous body colouration of golden yellow. Tabanus

par Walker is regarded as one of the most widely distributed Tabanus in sub-Saharan Africa

[40]. It has been reported in Senegal, Egypt, Mozambique, Congo, Ivory Coast, Zimbabwe,

Gambia and northern Nigeria [40]. Its abundance was noticed at the beginning of rains during

its collection in this study. It has similar features with T. thoracinus, which has its first report

from Benin Republic and well-distributed in other west African countries. This similarity

with other flies might have affected its reporting in the past, however, the transparent wing

pattern, shorter body length, tarsi differences and body width differentiates it from other flies.

In this study, T. par and T. thoracinus were found in Adebayo and UI, Oyo state and catches

were observed in wet and dry seasons. The short sequence obtained from T. par suggests that

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targeting other regions of the gene for identification and DNA barcoding of highly conserved

regions could be used in future studies. Due to the differences in nucleotides, the sequence of

this fly species was submitted to GenBank. Tabanus spp. found in Nigeria were similar to

those of Afrotropical region and genetically distinct from those in Neotropical or Nearctic

regions [57].

Stomoxys calcitrans is the most widely distributed in Africa and it has been reported from

Algeria and Egypt to Cape Colony (South Africa) and from Gambia to the East Africa

Protectorate (Kenya and Uganda) [40]. It has been reported in Europe, Middle East, South

America North America and New Zealand and it is regarded as a synanthropic fly [22] and

surveys in Nigeria suggest S. calcitrans is the most abundant Stomoxys spp. [29, 30, 42]. In

this study however, Stomoxys niger niger was most abundant among the trapped vectors and

distributed across the study sites. Stomoxys calcitrans was also trapped throughout the study

sites but with lower apparent density and low percentage catches in the dry season. The

observed differences between the two Stomoxys spp. could be attributed to climate change

and loss in biodiversity. The general reduction of Stomoxys spp. during heavy rainy months

could be due to washing away of larvae. The seasonal pattern and relative abundance

characterising all the studied vector species is a well-known phenomenon. Studies have

shown different vector species encountered seasonal fluctuations with increased catches at

the beginning of rains and decline along the dry season [58]. However, this is modulated by

regional climatic parameters. Environmental variables such as rainfall, temperature,

humidity, wind-speed and vegetation-type are major drivers of vector distribution and

abundance. The impact of these vectors on AAT and HAT infections could be overwhelming

[3, 5, 59, 60].

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In conclusion, proper identification of vectors is important in the surveillance of arthropod-

borne diseases. Hence, further studies on transmission patterns from these fly vectors to

livestock and humans will be important to understanding the epidemiology and management

of AAT.

Acknowledgements

We extend our sincere appreciation to livestock owners and support staff in southwest

Nigeria for their assistance during this study. This study was supported by Commonwealth

Scholarship Commission and The University of Edinburgh, United Kingdom. Paul O.

Odeniran is a Commonwealth scholar, funded by the UK government (reference number

NGCN-2016-196).

Ethical approval

The study was conducted with the permission of the University of Ibadan Animal Ethics

Committee (UI-ACUREC/App/12/2016/05) in line with the guidelines of the committee.

Conflict of interest

The authors declare that there is no conflict of interest.

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Figure captions

Fig. 1 Map showing trap locations of the study

Fig. 2 Phylogenetic analysis of Glossina species based on 28S ITS2 nuclear DNA in

southwest Nigeria. The taxa with GenBank accession no. represent the reference sequences

while those in red square brackets designated as GP1-13 =G. palpalis and GT1-7 =G.

tachinoides represent flies from this study.

Fig. 3 Median-neighbour joining networks of Palpalis group compared with other species of

Glossina from southwest Nigeria depicting interspecies variation and relatedness of the

representative haplotypes based on 28S ITS2 nuclear DNA. GF = Glossina fuscipes, GT = G.

tachinoides, GM =G. morsitans, GP =G. palpalis. Small black circles are median vectors i.e.

hypothetical of unsampled haplotypes.

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Fig. 4 Phylogenetic tree based on 28S ITS2 nuclear DNA of Tabanids showing the

relationship between species trapped in southwest Nigeria and flies from other geographical

location. Taxa labelled in red represent flies from this study.

Fig. 5 Phylogenetic tree based on 28S ITS2 nuclear DNA depicting the relationship between

Stomoxys species trapped in southwest Nigeria and flies from other geographical location.

SeqA-X in red square brackets represent flies from this study.

Table 1. Diversity and neutrality indices for Glossina palpalis and Glossina tachinoides populations based on partial 28S ITS-2 DNA gene

Indices G. palpalis (291 bp) G. tachinoides (291 bp)

Number of flies 13 7

Number of mutations 1 3

Parsimony informative sites 1 3

Number of haplotypes 2 2

Haplotype diversity (hd) 0.282±0.142 0.571±0.119

Nucleotide diversity (π) 0.00097 0.00589

Tajima’s d -0.27429 1.81122

Fu’s fs 0.240 2.920

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619

620

621

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Table 2. Fly species distribution and sex of captured Glossina spp., Tabanus spp., Ancala spp., Atylotus spp. and Stomoxys spp. flies in southwest Nigeria.

VECTOR VARIABLE LEVEL TOTAL % SPP. DIST.

95% CI

GLOSSINA SPECIES G. PALPALIS 64 47.4 39.2-55.8

G. TACHINOIDES

71 52.6 44.2-60.8

SEX F 72 53.3 44.9-61.5

M 63 46.7 38.5-55.1

TOTAL 135

TABANIDS SPECIES T. BIGUTTATUS 7 13.5 6.7-25.3

A. FASCIATA 4 7.7 3.0-18.2

T. GRATUS 2 3.8 1.1-13.0

T. PAR 5 9.6 4.2-20.6

T. PERTINENS 4 7.7 3.0-18.2

A. AGRESTIS 1 1.9 0.3-10.1

T. SUBANGUSTUS

11 21.2 12.2-34.0

T. TAENIOLA 15 28.8 18.3-42.3

T. THORACINUS 3 5.8 2.0-15.6

SEX F 42 80.8 68.1-89.2

M 10 19.2 10.8-31.9

TOTAL 52

STOMOXYINE SPECIES S. CALCITRANS 2402 27.6 26.8-28.4

S. NIGER NIGER 6295 72.4 71.4-73.3

SEX F 5253 60.4 59.4-61.4

M 3444 39.6 38.6-40.6

29

622623

624

5758

TOTAL 8697

30

625

5960

Table 3. Total catches of dipteran flies using Nzi traps in southwest Nigeria.

Fly species Season Sex Haematophagous TOTAL

Wet dry Male Female fed Unfed

Glossina palpalis (Vanderplank, 1911) 59 5 29 35 36 28 64

Glossina tachnoides (Westwood) 71 - 34 37 13 58 71

Tabanus taeniola (Pal.Beauvois) 13 2 3 12 11 4 15

Tabanus subangustus (Ricardo) 11 - 2 9 8 3 11

Tabanus biguttatus (Wiedemann) 6 1 1 6 5 2 7

Tabanus par (Walker) 4 1 2 3 2 3 5

Ancala fasciata (Fabricus) 3 1 - 4 3 1 4

Tabanus gratus (Loew) 2 - - 2 1 1 2

Atylotus agrestis (Wiedemann) - 1 - 1 1 - 1

Tabanus pertinens (Austen) 4 - 1 3 3 1 4

Tabanus thoracinus (Pal.Beauvois) 2 1 1 2 2 1 3

Chrysops silacea (Austen) 5 1 2 4 1 5 6

Chrysops longicornis (Macquart) 7 1 1 7 3 5 8

Chrysops distinctipennis (Austen) 1 - 1 - - 1 1

Haematopota pertinens (Austen) 2 - 2 - - 2 2

Stomoxys nigra (Macquart) 5259 1036 2388 3907 1934 4361 6295

Stomoxys calcitrans (Linnaeus) 2092 310 1056 1346 317 2085 2402

Simulium damnosum (Latreille) 15 - 4 11 4 11 15

Chrysomya putoria (Wiedemann) 15 4 2 17 - - 19

Chrysomya bezziana (Villeneuve) 1 - - 1 - - 1

Lucilia sericata (Meigen) 29 2 14 17 - - 31

Fannia canicularis (Linnaeus) 908 114 296 726 - - 1022

Fannia scalaris (Fabricius) 12 - - 12 - - 12

Hippobosca variegata (Megerle) 63 8 30 41 4 67 71

Musca domestica (Linnaeus) 3114 709 1680 2143 - - 3823

TOTAL 11698 2197 5548 8347 2348 6639 13895

31

626

627

6162

Figure 1

32

628

629630

631

632633

6364

Figure 2

33

634635

6566

Figure 3

34

636

637638639

6768

Figure 4

35

640641642

6970

Figure 5

36

643644645646

7172