Diversity Analysis of Adult Chironomidae in the Lake Victoria Basin of Kenya
41
Diversity Analysis of Adult Chironomidae in the Lake 
Victoria Basin of Kenya
Walter Odhiambo OTIENO
*
, Reuben Oyoo MOSI, Peter BULLI 
School of Agricultural and Food Sciences, Jaramogi Oginga Odinga University of Science and Technology,  
P. O. Box 210-40601, Bondo, Kenya
Agricultura Scientia 20: No 2: 41-53(2023)
https://doi.org/10.18690/agricsci.20.2.5
*Correspondence to: 
E-mail: walterodhiambo951@gmail.com
ABSTRACT
Chironomidae commonly inhabits most aquatic habitats and often dominate aquatic insect communities in abundance 
and species richness. Despite their ecological importance, the diversity and distribution of chironomids in the Lake Victoria 
ecosystem of Kenya have not been studied to date. Here we report on the diversity and distribution of adult Chironomidae in 
Usenge, Mbita and Ogal beaches of the Lake Victoria ecosystem in Kenya using morphological features and sequence data of 
Cytochrome c oxidase subunit I (COI) gene. Wing venation-based microscopic characterization identified four genera, Tanypus, 
Coelotanypus, Dicrotendipes and Chironomus. The COI gene barcoding further revealed several species, including Kiefferulus 
brevibucca, Chironomus flaviplumus, Polypedilum fuscovittatum, Polypedilum sp. and Dicrotendipes sp. The identified species 
were grouped into three clusters based on neighbor-joining phylogenetic approach. Differences in species richness were observed 
among the three study sites, with Mbita exhibiting the highest species richness. The evolutionary analysis revealed relatedness 
among all the identified species, suggesting a shared recent common ancestor. Unlike previous studies, this study represents the 
first report on detailed characterization of Chironomidae in the Lake Victoria ecosystem of Kenya. Moreover, this study serves as 
a first step towards a comprehensive understanding of the range of species of Chironomidae inhabiting this ecosystem.
Key words: Chironomidae, cytochrome c oxidase subunit I, DNA barcoding, wing venation, diversity
INTRODUCTION
Food demands of a rapidly growing world population (United 
Nations, 2017, Gu et al., 2021) are putting tremendous pressure 
on the available limited resources of water and land which 
also serve as important ecological habitats for many species. 
Climate change-driven variations in temperature and 
precipitation is further exacerbating the pressure on the 
limited resources through geographical shifts in habitats of 
many species. The impact of these challenges is pronounced 
in the Lake Victoria Basin of East Africa where human 
activities, coupled with climate change, have directly 
contributed to degradation of the lake and its surrounding 
terrestrial habitat, which is reflected in midge responses to 
these impacts (Nyakeya et al., 2018). The habitat degradation, 
which is reflected in the observed low genetic variation in 
species such as Nile perch (Lates niloticus) (Basiita et al., 2018) 
of the ecosystem, is worsening the already fragile balance 
of food webs in the lake ecosystem. For instance, changes 
in water quality and biological activities of the lake in the 
last five decades have caused shifts in populations of several 
species inhabiting the ecosystem, with the most notable 
change observed in the significant increase in biomass of 
chironomids which are commonly referred to as lake flies 
in the region (Gikuma-Njuru et al., 2005). Since ‘lake flies’ 
is a general term and can be misleading, we used the word 
‘chironomids’ throughout in this publication.
Chironomids, which belong to the order Diptera and the 
family Chironomidae, are an important food resource for 
fish, amphibians, dragonfly larvae, birds, bats, and spiders 
(Niemi et al., 1999, Wardiatno and Krisanti, 2013, Sharifian 
Fard et al., 2014). The chironomids are also an important 
tool in ecological and paleo-ecological studies, as well as in 
environmental evaluation, agricultural entomology, and 
public-health research (Rosenberg, 1992, Ferrarese, 1992, 
Armitage et al., 1995). Although not widely reported, the chi-
ronomids are also one of the commonly consumed insects by 
the communities inhabiting the region in addition to their 

Diversity Analysis of Adult Chironomidae in the Lake Victoria Basin of Kenya
42
ecological importance (Ayieko and Oriaro, 2008). Recently, 
the chironomids are sighted less frequently (Ayieko et al., 
2010b), raising fears among the local communities of decline 
in their population. In spite of the importance of chirono-
mids as link between the aquatic and terrestrial food web 
(McCary et al., 2021), and as sources of protein to the local 
community (Ayieko and Oriaro, 2008), their diversity and 
distribution around the region has not been studied to date. 
Knowledge on the diversity of this important component of 
ecosystem will not only greatly enhance our understand-
ing of the fauna and ecology of Lake Victoria, but will also 
enable formulation of evidence-based decisions on conser-
vation of their biodiversity considering that the predicted 
exponential growth of human population in the region is 
expected to lead to increased consumption of the indigenous 
insects (Ayieko et al., 2010a). Therefore, to further enhance 
our understanding of the fauna and ecosystem of Lake 
Victoria, and contribute to the national and global database 
of Chironomidae in the region, this study is designed to 
characterize the diversity of Chironomidae in different sites 
of Lake Victoria.
MATERIAL AND METHODS
Study Area
The Lake Victoria Basin of Kenya (LVB) is located in the 
western part of the country (Fig. 1) with an approximate 
area of 4,113 km
2
. The lake lies at an altitude of 1,134 m above 
sea level, and lies between latitudes 0
o
 20’ N, 3
o
 0’ S and 
longitudes 31
o
 39’ E, 34
o
 53’ E. Lake Victoria, the most notable 
landmark of the region, has an average depth of approx. 40 
m, with a maximum depth of 84 m. LVB comprises seven 
districts, including parts of Siaya, Bondo, Kisumu, Nyando, 
Rachuonyo, Homa Bay, Migori, Suba and Busia, with a 
population of over 4 million people. Diverse ethnic groups 
of indigenous people sharing similar livelihoods within 
the region include the Luo, Luhya, Kisii, Kuria, Suba, Teso, 
Kalenjin and Maasai. The main economic activities in the 
lake region are fishing, farming, bee keeping, sand and gold 
mining and trading (UNEP, 2006).
Morphological Characterization of Chironomidae in 
the Lake Victoria Basin of Kenya
Sampling of Chironomidae
Adult chironomids were collected from three sites: Usenge 
Beach (latitude 0
o
 24’ 54.7848” N; longitude 34
o
 12’ 14.3136” E), 
Mbita (latitude 0
o
 4’ 20.28” N; longitude 34
o
 3’ 39.4848” E) and 
Ogal Beach (latitude 0
o
 8’ 27.3264” N; longitude 34
o
 35’ 36.1428” E) 
in Siaya, Homabay and Kisumu Counties, respectively. The 
sites were selected based on frequent seasonal sightings of the 
chironomids. Samples of chironomids were collected twice, 
December 2020 to January 2021, and June 2021, coinciding 
with their seasonal emergence. Flying chironomids were 
trapped using a sweep net comprising a net tied to a round 
metallic ring and a 1.5 m-long wooden handle. To trap 
 
 
Figure 1: Map of Lake Victoria Basin, Kenya

Diversity Analysis of Adult Chironomidae in the Lake Victoria Basin of Kenya
43
the flies, the net was whirled in the air. Shrubs and trees, 
along the shores of the lake, were occasionally shaken to 
disturb the flies and force them to fly during collection as 
described by Ayieko and Oriaro (2008). Collected samples 
were preserved in 70% ethanol in plastic containers, and 
transported to the laboratory for sorting and subsequent 
studies. For each collection site, there were nine samples 
(with each containing an average of approx. 151 flies), giving a 
total of 54 samples for the two sampling periods of December 
2020 to January 2021, and June 2021. 
Morphological characterization
Morphological characterization was performed at the 
National Museums of Kenya. Wings were dissected using 
fine needles under a dissecting Wild M3 microscope (Wild 
Heerbrugg Co., Switzerland), and then mounted on semi-
permanent slides for characterization to group them into 
genera. Morphological characters that aided identification 
were first branch of anal vein (A
1
); basal-medial crossvein 
(bm-m); costal vein (C); calypter (calyp); cubitus (Cu); 
anterior branch of cubital vein (CuA); cubital vein fork 
(Cu-fork); posterior branch of cubital vein (CuP); media (M); 
fused first and second branch of media (M
1+2
); fused third 
and fourth branch of media (M
3+4
); medial-cubital crossvein 
(m-cu); anterior branch of radius (R
1
); upper branch of 
second branch of radius (R
2
); second branch of radius (R
2+3
); 
lower branch of second branch of radius (R
3
); third branch of 
radius (R
4+5
); and radial-medial crossvein (r-m) (Kirk-Springs 
and Sinclair, 2017).
Diversity analysis
The diversity indices of Shannon (H’) and evenness index 
were used to assess insect diversity within and between 
sites. Shannon-Wiener diversity index Hp ip i
i
s
'
ln 


1
; where s = number of genera; and p
i
 = proportion of the 
i-th genus, n
i
/N. Shannon evenness = 
H
s
'
ln
  where s = total 
number of genera. The Dominance index (D = P
max
) was used 
to express the proportion of individuals accounted for by 
the most abundant genera in each site; where P
max
 is the 
maximum proportion of any one genus in a sample. All the 
above diversity indices were performed using PAST Software 
Version 4.10 (https://www.nhm.uio.no/english/research/
infrastructure/past/). 
Molecular Characterization of Chironomidae
Genomic DNA extraction
To help support taxonomic identifications, along with 
having a greater chance of determining novel taxa that 
were not identified by the microscopic-based morphological 
characterization, the specimens were separated into 
taxonomic groups based on morphology and then specimens 
were randomly selected from within those groups. Total 
genomic DNA was extracted from 4 of the random samples 
per site using AuPrep Genomic DNA Extraction Kit (Life 
Technologies, Deli, India) following the manufacturer’s 
instructions. Each sample was ground and placed into 1.5 
ml sterile tubes. 180 µl LYS buffer was added to each sample 
followed by 20 µl Proteinase K. The samples were mixed by 
vortexing for 20 s. To lyse the samples, they were incubated 
at 60
o
 C for 1 hr 300 µl FX buffer was added to the samples 
and mixed by vortexing, followed by incubation at 70
o
 C 
for 20 min. Two hundred microliter of 98% ethanol was 
added to the samples, followed by vortexing. The mix was 
pipetted into a B/T Genomic DNA Mini column placed onto 
a Collection Tube; and centrifuged at 8,000 rpm (6,000 xg) for 
2 mins. The column was then placed onto a new Collection 
Tube. The column was washed twice with 0.5 ml WS Buffer 
by centrifuging at 8,000 rpm for 2 min. The column was 
centrifuged at full speed (10,000 xg) for another 2 mins to 
remove ethanol residue, and then placed onto a new 1.5 ml 
tube. DNA was eluted with 200 µl of the preheated elution 
solution. The column was left to stand for 5 mins, and then 
centrifuged for 2 mins to elute DNA. The extracted DNA was 
quantified, subjected to quality analysis, and stored at -20
o
 C.
Amplification of cytochrome c oxidase subunit I gene
The chironomid COI barcode approach (Kang et al., 2022, Vasquez et 
al., 2022) is widely and effectively used to characterize chironomids 
at the species level. Cytochrome C Oxidase Subunit I (COI) gene 
was amplified following established protocols using the pairs of 
primers, LCO1490: 5’-GGTCAACAAATCATAAAGATATTGG-3’ and 
HCO2198: 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’ as 
described by Kim et al. (2012); and TL2-N-3014 (Pat): 
5’-TTCAATGCACTTATTCTGCCATATTA-3’) and C1-J-2138 (Jerry): 
(5’-CAACATTTATTTTGATTTTTTGG-3’). Briefly, the PCR reaction 
mix included 1 µl of forward primer, 1 µl of reverse primer, 1 µl 
of 10 ng genomic DNA templates, 10 µl of OneTaq 2xMaster Mix 
with standard buffer, and 7 µl of nuclease free water. To amplify 
the 5’ region of mitochondrial COI gene, the following thermal 
cycling program was used: hot start at 94 
o
C for 5 mins, 35 cycles 
of 94 
o
C for 30 s, 50 
o
C for 30 s and 68 
o
C for 60 s, followed by a 
final extension at 68 
o
C for 10 mins. The PCR products were held 
at 4 
o
C. Integrity of the PCR amplicons was visualized on a 1% 
CSL-AG500 agarose gel (Cleaver Scientific Ltd, UK) stained with 
EZ-vision Bluelight DNA Dye. 
Purification of PCR product
PCR products were purified using ExoSAP-IT™ PCR Product 
Cleanup Reagent (ThermoFisher Scientific, MA, USA) 
following the manufacturer’s protocol. ExoSAP master mix 
was prepared from 50 µl of Exonuclease I and 200 µl of Shrimp 

Diversity Analysis of Adult Chironomidae in the Lake Victoria Basin of Kenya
44
Tamura et al., 2021) with the following default settings: 
gap open -400.00; gap extend 0.00; maximum memory 
2,048 megabytes; maximum iterations 16; cluster method 
(iterations 1,2) UPGMA; cluster methods (other iterations) 
UPGMA; and minimum diagonal length (lambda) 24. 
Substitution model for genetic analysis
Kimura-2 parameter (K2P) distance model (Kimura, 1980) was 
used for calculating intraspecies and interspecies sequence 
divergences, pairwise distances and overall distance. 
Assuming P is the fraction of nucleotide sites that show Type 
I difference or transition type (homologous sites occupied by 
different nucleotide bases but both are purines or both are 
pyrimidines), and Q the fraction of nucleotide sites showing 
Type II difference or transversion type (one of the two 
nucleotide bases is a purine and the other is a pyrimidine) 
between two sequences, the evolutionary distance per site is 
calculated using the formula KP QQ 






  




1
2
12 12 ln . 
The evolutionary rate per year is given by k=K/(2T); where T = 
time since the divergence of the two sequences. 
Evolutionary Analysis
Disparity index test (Kumar & Gadagkar, 2001) was used 
to test the homogeneity of substitution patterns between 
the 11 sequences. Pairwise and within group distances 
were estimated using Kimura 2-parameter model. Monte 
Carlo test of 1,000 replicates to estimate the P-values and 
standard errors. Ambiguous positions were removed for 
each sequence pair (pairwise deletion option). P-values 
smaller than 0.05 are considered significant. The analysis 
was performed for codon positions 1st+2nd+3rd+Noncoding. 
A total of 730 positions were included in the final dataset. 
All the evolutionary analyses were conducted using MEGA 11. 
The number of base substitutions per site from averag-
ing over all sequence pairs within and between sites were 
shown. Standard error (SE) estimates were obtained by a 
bootstrap procedure of 1000 replicates. Analyses were con-
ducted using the Kimura 2-parameter model. This analysis 
involved 11 nucleotide sequences. Codon positions included 
were 1st+2nd+3rd+Noncoding. All ambiguous positions were 
removed for each sequence pair (pairwise deletion option). 
There was a total of 730 positions in the final dataset.
Neighbor-joining statistical method was used to construct 
phylogenetic tree based on the 11 nucleotide sequences of the 
COI gene fragments amplified from DNA of randomly selected 
chironomids from the three sites using MEGA version 11.0.13. 
Bootstrap was 1,000 replications with the following settings: 
substitution model Kimura 2-parameter, substitutions in-
cluding transitions and transversions, uniform rates among 
sites, homogenous pattern among lineages, pairwise deletion 
for gaps removal, and 1
st
, 2
nd
 and 3
rd
 codon positions, and non-
coding sites selected.
Alkaline Phosphatase in a 0.6 ml micro-centrifuge tube. Ten 
microliters (10 µl) of post-PCR reaction product was mixed 
with 2.5 µl ExoSAP Mix. The reaction mixture was well mixed 
and incubated at 37 
o
C for 15 mins to degrade remaining 
primers and nucleotides. To inactivate the ExoSAP-IT reagent, 
the mix was incubated at 80 
o
C for 15 mins. The purified PCR 
products were stored at -20 
o
C prior to sequencing.
Sequencing of PCR products
Amplified DNA fragments were sequenced using the 
BrilliantDyeTM Terminator (v3.1) Cycle sequencing Kit 
(NimaGen B.V., The Netherlands) according to manufacturer’s 
instructions. For labelling of the amplified PCR fragments, 
the cycle sequencing reaction contained 1 µl BrilliantDye 
v1.1 rr Premix, 3.5 µl 5x sequencing buffer, 1 µl template, 1 µl 
primer (3.2 - 5 pMol), and 13.5 µl water. The thermal cycling 
protocol was as follows: initial denaturation of 96 
o
C for 45 
s, followed by 25 cycles of 96 
o
C for 10 s, 50 
o
C for 5 s and 
60 
o
C for 4 mins. Before capillary electrophoresis the cycle 
sequencing products were purified to remove unincorporated 
fluorescent ddNTPs and salts using ZR-96 DNA Sequencing 
Clean-up Kit (Zymo Research Corporation, USA). The purified 
products were injected on an Applied Biosystems ABI 3500XL 
Genetic Analyzer (ThermoFisher Scientific Inc, USA) with a 
50 cm array, using POP7. For control, 1 µL of pGEM plasmid 
DNA template and 1 µL of -21M13 primer provided in the 
BrilliantDyeTM Terminator (v3.1) Cycle sequencing Kit 
were included in the sequencing reaction to verify the 
performance of the total workflow and troubleshooting 
correlated to the amplified COI gene fragments and primers. 
Raw sequences were edited using BioEdit 7.2 software (Hall 
et al., 2011) to remove the ‘N’s and trim the beginning and 
end of the sequences to maintain high accuracy. Consensus 
sequences were derived from forward and reverse sequences.
Analysis of sequences of COI fragments
Sequence similarity search
Megablast program of the Nucleotide Basic Local Alignment 
Search Tool (BLASTn) was used to search for similarities 
between sequences of the amplified COI fragments and 
sequences in the Genome (NIH_TR_1.0 reference Annotation 
release 100) database of the National Center for Biotechnology 
Information (NCBI). Cut off for similarity between sequences 
in the database and the COI query sequences was 80%.
Multiple sequence alignment
Sequences of the COI fragments were aligned with their 
corresponding accessions retrieved from the NCBI database 
using MUSCLE (Multiple Sequence Comparison by Log-
Expectation) tool in the software MEGA 11 (Edgar, 2004, 

Diversity Analysis of Adult Chironomidae in the Lake Victoria Basin of Kenya
45
 
 Figure 2: Wing venation: (A) Tanypus; (B) Coelotanypus; (C) Dicrotendipes; and (D) Chironomus
RESULTS
A total of 8,161 individual Chironomidae from 54 samples 
were characterized. The average number of flies per sample 
was approximately 151 (Table 1). In total, 4 genera, Tanypus, 
Coelotanypus, Dicrotendipes and Chironomus were identified 
based on wing venations using a dissecting microscope (Fig. 
2a-d). The wing of Coelotanypus is characterized with vein R
2 
(upper branch of second branch of radius) not connected to 
vein R
3
 (lower branch of second branch of radius); while the 
wing of Tanypus was characterized with a distance between 
Cu-fork (cubital vein fork) and cross-vein of less than a 
third the length of vein CuA (anterior branch of cubital 
vein). Both Dicrotendipes and Chironomus have wings with 
cross-vein r-m (radial-medial crossvein) oblique to vein R
4+5
 
(third branch of radius). The number of Chironomidae per 
genus and their percent distribution across the three study 
sites are shown in Table 1 and Fig. 3a-c. At the taxonomic 
level, Dicrotendipes and Chironomus were the only 
genera identified in Usenge, with Dicrotendipes being the 
predominant genus. Three genera were identified at Ogal, 
represented with 54, 39 and 7% of Tanypus, Coelotanypus and 
Chironomus. At Mbita site, all four genera were identified, 
with Tanypus, Coelotanypus, Dicrotendipes and Chironomus 
accounting for 9, 8, 59 and 24% of the collected samples of 
Chironomidae. Chironomus was the only genus collected 
from all three sites.
Table 1: Chironomidae collected from sampling sites
Genus/Site Usenge beach Mbita Ogal beach Total
Tanypus 0 124 1730 1854
Coelotanypus 0 112 1265 1377
Dicrotendipes 2312 804 0 3116
Chironomus 1268 331 215 1814
Total 3580 1371 3210 8161

Diversity Analysis of Adult Chironomidae in the Lake Victoria Basin of Kenya
46
Diversity analysis
The diversity indices are summarized in Table 2. All four 
genera were recorded in Mbita, with Dicrotendipes being 
the most abundant. An uneven distribution of the number 
of genera was observed in Mbita, with Dicrotendipes being 
the dominant genus (Figure 3). Similarly, the Shannon 
diversity index revealed a relatively higher level of diversity 
in Mbita, followed by Ogal and lastly Usenge. The individual 
Chironomidae for the four genera in Mbita are more evenly 
distributed compared to those of both Usenge and Ogal.
Table 2: Diversity indices for Chironomidae genera collected 
at Lake Victoria
Parameter
Indices
Usenge Mbita Ogal
Taxa S (Genera) 2
b
4
a
3
ab
Individuals 3580
a
1371
c
3210
b
Dominance D 0.5425
a
0.417
c
0.4502
b
Shannon H 0.65
c
1.078
a
0.8812
b
Evenness 0.0794
c
0.1492
a
0.1091
b
Values with different superscripts in rows are significantly different at p < 0.05
DNA Barcode analysis
Amplified fragments using primers targeting the COI gene 
region from 12 random samples of individual Chironomidae 
were sequenced (hereafter referred to as Seq #1 to Seq #12). 
Seq #8 was excluded from further analysis because the quality 
of the reverse sequence was poor. Sequence similarity search 
was performed using the NCBI Blastn database, and the 
results are shown in Table 3. The length of the 11 sequences 
ranged from 466 to 678 base pairs. The low Expected values 
(e-values) for all the searches indicated very high scores (S) 
for the matches between the queries and sequences in the 
NCBI database, with 96-100% of the query lengths included in 
the aligned sequences. The percent similarity ranged from 
83.6 to 94.7. The sequence similarity search identified a total 
of 5 species, including Kiefferulus brevibucca, Chironomus 
flaviplumus, Polypedilum fuscovittatum, Polypedilum sp. 
and Dicrotendipes sp. Kiefferulus brevibucca was identified 
in Ogal and Mbita; Chironomus flaviplumus and Polypedilum 
fuscovittatum were identified in Usenge and Mbita; and 
Polypedilum sp., and Dicrotendipes sp. were identified in 
Ogal. The four sequences labeled Chironomidae could likely 
represent unknown or poorly sequenced genera since they 
are not identified at a finer resolution than family.
Evolutionary relationships of taxa
The evolutionary history was inferred using the Neighbor-
Joining method (Saitou & Nei, 1987). The bootstrap consensus 
tree inferred from 1,000 replicates (Felsenstein, 1985) is 
taken to represent the evolutionary history of the taxa 
analyzed. Branches corresponding to partitions reproduced 
in less than 50% bootstrap replicates are collapsed. The 
evolutionary distances were computed using the Kimura 
2-parameter method (Kimura, 1980) and are in the units 
of the number of base substitutions per site. Ambiguous 
positions were removed for each sequence pair (pairwise 
deletion option). The final dataset included a total of 
730 positions. Three major clusters were identified (Fig. 
4). Cluster 1 is the largest, and is comprised of Kiefferulus 
brevibucca, Dicrotendipes sp., Polypedilum sp., Polypedilum 
fuscovittatum, and Chironomidae sp. Cluster 2 consists of 
Chironomidae sp. (Sequence 10), Chironomidae sp. (Sequence 
2) and Chironomidae sp. (Sequence 7); while Chironomus 
flaviplumus are grouped separately into Cluster 3. The 
 
 
Figure 3: Distribution of Chironomidae genera among the three sites: Usenge (a), Mbita (b) and Ogal (c)

Diversity Analysis of Adult Chironomidae in the Lake Victoria Basin of Kenya
47
species in each of Clusters 1 and 2 are from the three study 
sites of Mbita, Ogal and Usenge. The sequences for Kiefferulus 
brevibucca from Ogal and Mbita are identical. Similarly, 
Chironomus flaviplumus clusters with Chironomus 
flaviplumus, both collected from Usenge. The Chironomidae 
sp. collected from Ogal and Chironomidae sp. collected from 
Mbita also cluster together suggesting that they share the 
most recent common ancestor at branch point with 56%. 
Three Chironomidae spp. share the most recent common 
ancestor at the node with 99%. Polypedilum fuscovittatum 
from Mbita and Chironomidae sp. from Usenge (Sequence 9) 
are distantly related as the Kiefferulus brevibucca from Ogal 
and Mbita based on the genetic distance between them. 
Evolutionary Relationships Between Species
The Disparity Index tests the null hypothesis that sequences 
have evolved with the same pattern of substitution, as 
judged from the extent of differences in base composition 
biases between sequences (Kumar & Gadagkar, 2001). It is the 
probability of rejecting the null hypothesis that sequences 
have evolved with the same pattern of substitution. A Monte 
Table 3: NCBI Blast hit for partial gene sequences of COI amplified from samples of Chironomidae
Site
Query Blast output
Name
Length 
(bp)
Species
Max 
score
Total 
score
Query 
cover (%)
E-value
Similarity 
(%)
Accession 
length (bp)
Accession
Ogal
Seq #1 550 Polypedilum sp. 701 701 100 0.0 89.66 658 KU497053.1
Seq #2 582 Chironomidae 760 760 99 0.0 90.19 645 MT534793.1
Seq#3 609 Kiefferulus brevibucca 942 942 99 0.0 94.72 663 DQ648215.1
Seq #4 597 Dicrotendipes sp. 743 743 100 0.0 89.15 657 JF286810.1
Mbita
Seq #5 622 Kiefferulus brevibucca 952 952 97 0.0 94.58 663 DQ648215.1
Seq #6 588 Polypedilum fuscovittatum 754 754 100 0.0 89.80 658 LC329185.1
Seq #7 592 Chironomidae 774 774 99 0.0 90.48 645 MT534793.1
Usenge
Seq #9 466 Chironomidae 424 424 96 0.0 83.63 579 KR512199.1
Seq #10 513
Chironomidae 682 682 99 0.0 90.23 640 MT535262.1
Chironomidae 682 682 99 0.0 90.23 640 MT534815.1
Chironomidae 682 682 99 0.0 90.23 645 MT534793.1
Chironomidae 682 682 99 0.0 90.23 648 MT534749.1
Chironomidae 682 682 99 0.0 90.23 638 MT534748.1
Seq #11 678 Chironomus flaviplumus 815 815 98 0.0 88.47 823 MN968308.1
Seq #12 663 Chironomus flaviplumus 785 785 99 0.0 87.84 823 MN968308.1
 
 
 Kiefferulus brevibucca (Ogal 3)
 Kiefferulus brevibucca (Mbita 5)
 Dicrotendipes sp. (Ogal 4)
 Polypedilum sp. (Ogal 1)
 Polypedilum fuscovittatum (Mbita 6)
 Chironomidae sp. (Usenge 9)
 Chironomidae sp. (Usenge 10)
 Chironomidae sp. (Ogal 2)
 Chironomidae sp. (Mbita 7)
 Chironomus flaviplumus (Usenge 11)
 Chironomus flaviplumus (Usenge 12)
100
100
56
99
54
51
69
43
Figure 4: Neighbor-Joining phylogenetic tree inferred from COI fragments. The percentage of replicate trees in which the 
associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches

Diversity Analysis of Adult Chironomidae in the Lake Victoria Basin of Kenya
48
Carlo test with 1000 replicates was used to estimate the 
p-values, with values less than 0.05 considered significant 
(Table 4). The number of base substitutions per site from 
between sequences are shown (Table 5) and were obtained 
by a bootstrap procedure (1000 replicates). Analyses were 
conducted using the Kimura 2-parameter model. This 
analysis involved 11 nucleotide sequences. Codon positions 
included were 1st+2nd+3rd+Noncoding. All ambiguous 
positions were removed for each sequence pair (pairwise 
deletion option). There was a total of 730 positions in the final 
dataset. Significant differences in patterns of substitution 
were observed in 26 out of 55 pairwise comparisons (Table 
4), implying that the evolutionary process in close to half 
of the pairwise comparisons. The Polypedilum sp. from 
Ogal is distantly related to the Chironomidae sp. from Ogal 
and Usenge, respectively; while the evolutionary pattern 
of the Chironomidae sp. from Ogal is similar to that of 
the Chironomidae sp. and Chironomus flaviplumus from 
Usenge. There are no significant differences in evolutionary 
patterns among Dicrotendipes sp, Kiefferulus brevibucca, 
Chironomidae sp, and Chironomus flaviplumus from Ogal, 
Mbita, and Usenge, respectively. With the exceptions of the 
Chironomidae sp. from Ogal and Polypedilum fuscovittatum 
from Mbita, there were no significant differences in 
evolutionary patterns among sequences of Polypedilum 
sp, Kiefferulus brevibucca and Dicrotendipes sp. from Ogal, 
Kiefferulus brevibucca and Chironomidae sp. from Mbita, 
and the Chironomidae sp. and Chironomus flaviplumus from 
Usenge. Kiefferulus brevibucca from Mbita is distantly related 
to the Chironomidae sp. and Chironomus flaviplumus. The 
evolutionary pattern of Polypedilum fuscovittatum from 
Mbita significantly differs from that of Chironomidae sp. 
from Mbita and Usenge. There are no significant differences 
in evolutionary pattern among the Chironomidae sp. from 
Mbita, Polypedilum sp. from Ogal, and Chironomidae sp. and 
Chironomus flaviplumus from Usenge. The Chironomidae 
sp. from Usenge is distantly related to the Chironomidae sp. 
and Chironomus flaviplumus from Usenge. The evolutionary 
patterns of the Chironomidae sp. and that of Chironomus 
flaviplumus from Usenge are similar. The Chironomus 
flaviplumus, from Usenge share similar evolutionary patterns. 
Results from the rest of the homogeneity of substitution 
patterns between the identified species is further validated 
by the estimates of evolutionary distances between the 
species.
Estimates of Average Evolutionary Divergence
The average intraspecific divergence was 31.3% (Table 6). 
The highest rate of intraspecific diversity was observed in 
Usenge where three distinct species, including two species of 
Chironomidae and Chironomus flaviplumus were identified. 
Table 4: Test of the homogeneity of substitution patterns between sequences
Site Seq # Blast hit 1 2 3 4 5 6 7 9 10 11 12
Ogal 1 Polypedilum sp.
Ogal 2 Chironomidae sp. 0.030
*
Ogal 3 Kiefferulus brevibucca 0.118
NS
0.001
**
Ogal 4 Dicrotendipes sp. 1.000
NS
0.006
**
1.000
NS
Mbita 5 Kiefferulus brevibucca 0.118
NS
0.001
**
1.000
NS
1.000
NS
Mbita 6 Polypedilum fuscovittatum 1.000
NS
0.003
**
0.033
*
0.140
NS
0.018
*
Mbita 7 Chironomidae sp. 0.066
NS
0.010
*
0.005
*
0.027
*
0.002
**
0.000
***
Usenge 9 Chironomidae sp. 0.112
NS
0.000
***
1.000
NS
0.050
NS
1.000
NS
0.001
**
0.005
**
Usenge 10 Chironomidae sp. 0.003
**
1.000
NS
0.024
*
0.107
NS
0.047
*
0.009
**
0.111
NS
0.000
***
Usenge 11 Chironomus flaviplumus 0.163
NS
0.033
*
0.042
*
0.123
NS
0.038
*
1.000
NS
0.041
*
0.008
**
0.145
NS
Usenge 12 Chironomus flaviplumus 0.247
NS
0.068
NS
0.063
NS
0.180
NS
0.054
NS
1.000
NS
0.065
NS
0.013
*
0.184
NS
1.000
NS
*, ** and *** = significant values at p < 0.05, p < 0.01, and p < 0.001; NS = non-significant value
Table 5: Estimates of evolutionary distances between species
Site Seq # Blast hit 1 2 3 4 5 6 7 9 10 11 12
Ogal 1 Polypedilum sp.
Ogal 2 Chironomidae sp. 0.160
Ogal 3 Kiefferulus brevibucca 0.138 0.239
Ogal 4 Dicrotendipes sp. 0.139 0.206 0.151
Mbita 5 Kiefferulus brevibucca 0.129 0.232 0.002 0.140
Mbita 6 Polypedilum fuscovittatum 0.163 0.208 0.163 0.176 0.160
Mbita 7 Chironomidae sp. 0.165 0.005 0.237 0.214 0.228 0.202
Usenge 9 Chironomidae sp. 0.250 0.241 0.322 0.288 0.310 0.253 0.239
Usenge 10 Chironomidae sp. 0.173 0.002 0.248 0.214 0.237 0.205 0.006 0.232
Usenge 11 Chironomus flaviplumus 0.776 0.777 0.833 0.791 0.830 0.821 0.779 0.862 0.737
Usenge 12 Chironomus flaviplumus 0.772 0.765 0.822 0.780 0.815 0.814 0.772 0.866 0.733 0.002

Diversity Analysis of Adult Chironomidae in the Lake Victoria Basin of Kenya
49
The species in Mbita and Ogal, respectively, exhibited 
relatively lower levels of intraspecific diversity. We observed 
relatively higher levels of genetic dissimilarities between 
species in Ogal and Usenge, and between species in Mbita and 
Usenge, respectively. Overall, a moderately low evolutionary 
divergence (d = 0.38 with a standard error of 0.02) was observed 
over all the species identified in the three sites.
Table 6: Estimates of average evolutionary divergence
Site Within sites (d)
Between sites
Ogal Mbita Usenge
Ogal 0.17±0.01 0.012 0.031
Mbita 0.20±0.02 0.153 0.031
Usenge 0.57±0.05 0.503 0.507
Values above the diagonal (between sites) are standard error estimates
DISCUSSION
Characterization of Chironomidae
Chironomids (Diptera: Chironomidae) are the most globally 
diverse and widely distributed aquatic insects. Despite 
their presence in the Lake Victoria ecosystem of Kenya, 
little is known about the diversity of chironomids relative 
to other aquatic insect taxa. The morphological-based 
characterization using wing venations identified only 
four genera, Tanypus, Coelotanypus, Dicrotendipes and 
Chironomus from the many specimens. Complementing the 
morphological approach with analysis of the Cytochrome c 
oxidase subunit I sequence fragment yielded more detailed 
results, identifying eight groups at the generic level, 
and 5 at the species level. The molecular results clearly 
suggest the presence of many more taxa occurring in the 
samples which were likely masked by Dicrotendipes and 
Chironomus. Although the DNA barcode data has been 
extremely important to this study, our work likely scratched 
the surface of diversity of the chironomids as we used a 
relatively smaller sample size for sequencing. DNA barcode 
data from larger samples of chironomids collected from the 
shores of the lake as well as adjacent habitats using sweep 
nets could have yielded a more detailed and comprehensive 
information on the diversity. The usefulness of integrating 
DNA barcoding with morphological-based characterization 
of chironomids was demonstrated by Stur and Ekrem 
(2020) who could only detect morphological differences of 
Chironomidae of Svalbard and Jan Mayen to closely related 
species after DNA barcodes indicated distinct genetic lineage. 
Diversity of Chironomidae in the Lake Victoria
The most widespread genus, occurring in all three study 
sites, was Chironomus, a genus widely distributed worldwide 
with 19 species reported in the Afrotropical Region (Kirk-
Spriggs & Sinclair, 2017). Eggermont et al. (2005) also reported 
five morphotypes of Chironomus larvae in the East African 
lakes. Ayieko & Oriaro (2008) previously reported Chironomus 
as the only genus in the Lake Victoria region of Kenya 
using personal observation methods and non-structured 
interviews with relevant local authorities. The findings 
reported by Ayieko and Oriaro (2008) clearly demonstrated 
the limitation of morphological characterization based 
on visual observation in identification of Chironomidae 
at the species level. They used color as a morphological 
marker, with black colored insects characterized as 
Chironomus. Additionally, the information collected from 
the local community leaders was likely biased as the local 
community residing in the region commonly refer to all 
insects resembling the chironomids as ‘kitambo’ in the 
local language. Moreover, the use of wing vein patterns-
based characterization in this study may have also created 
a situation where specimens were lumped into a limited 
number of categories that are not always accurate. Although 
Ayieko and Oriaro (2008) published a very interesting 
paper, they approaching their study of chironomids of 
the Lake Victoria region as agriculturalist and/ or cultural 
anthropologists rather not biologist or taxonomists. Thus, 
as interesting as their findings are to the growing literature 
on chironomids, they have a very limited influence on our 
taxonomic work. As suggested by Stur and Ekrem (2020), 
suitable identification keys for specific regions are essential 
for correct identification of chironomids to species using 
morphology since reliance on identification keys designed 
for other geographical regions is prone to errors.
Although the microscopic-based morphological 
approach identified the genera Coelotanypus and Tanypus, 
analysis of the sequence data did not reveal the two genera. 
However, this does not rule out their presence as the 
samples collected for sequencing of the COI region are likely 
not representative of all species of the flies inhabiting the 
shores of the lake. Twenty-one species of Coelotanypus have 
been reported, with some found in the Afrotropical Region. 
The genus Tanypus is characterized with relatively fewer 
species (only five have been reported so far) (Kirk-Spriggs 
& Sinclair, 2017). In addition to Chironomus, Coelotanypus 
and Tanypus, species of the genus Dicrotendipes identified 
in this study through both the morphological and molec-
ular-based approaches, have also been previously reported 
in the Afrotropical Region. Other species identified in the 
Lake Victoria region based on sequence information of the 
COI region include Polypedilum sp., Kiefferulus brevibucca , 
Polypedilum fuscovittatum and Chironomus flaviplumus . 
The presence of K. brevibucca is highly likely given that it 
has been reported previously in some parts of sub-Saharan 
Africa (Cranston and Martin, 1989). Moreover, K. brevibucca, 
P. fuscovittatum and C. flaviplumus are known from the 
Palearctic region (i.e. the biogeographic region that includes 
Europe, Asia north of the Himalayas, and Africa north of the 
Sahara), and thus may represent similar African species that 

Diversity Analysis of Adult Chironomidae in the Lake Victoria Basin of Kenya
50
are not yet described or are not yet separable from similar 
species. Based on the test of the homogeneity of substitution 
patterns between sequences, there are likely two different 
species of Chironomidae, with the two occurring in Usenge 
beach, and one each in the other two sites.
Evolutionary relationship among species of 
Chironomidae
Mitochondrial genomes (mitogenomes) have been widely 
used for studying the taxonomy and phylogeny of insects (Li 
et al., 2022). One mitochondrial gene, Cytochrome c oxidase 
subunit I (COI), was as phylogenetic marker in this study 
to provide an insight into their evolutionary relationships. 
The nucleotide sequence of 596 bp from COI was used to 
determine 11 DNA samples of 11 chironomids collected from 
Usenge, Mbita and Ogal beaches of Lake Victoria; and the 
nucleotide sequence alignments were used for construction of 
phylogenetic trees based on neighbor-joining methods. One 
key finding is that sampling site is not important in grouping 
the species into clusters. Rather, the grouping of species from 
the different sites appeared random. A likely explanation 
is that there is limited environmental variations among 
the three sites. In addition, grouping of different species 
into the same cluster groups is an indication of a shared 
common ancestor among the species, a fact supported by 
similarities in nucleotide substitution rate between species, 
and low intraspecific divergence. An important remaining 
question is the determination of the position of Chironomus 
flaviplumus in relation to the other species of the identified 
Chironomidae. C. flaviplumus (probably Palearctic) is a global 
species complex, and the species in Lake Victoria is likely a 
similar but inseparable species.
CONCLUSION
In this study, we found more diversity among chironomids 
in Lake Victoria Region of Kenya than previously reported. 
The use of microscopic-based morphological and molecular-
based approaches allowed us to point out the limitation 
of the former which is only efficient at the genus level 
of identification. However, in spite of the limitations of 
the morphological-based characterization, there are still 
excellent keys to the species level that encompass a majority 
of the diversity present in many bio-regions. The DNA 
barcoding using Cytochrome c oxidase subunit I (COI) gene 
as a marker revealed 5 species of the chironomids along the 
shores of the Lake Victoria. Although the number of the 
species identified is relatively smaller, it is an important 
start to what is likely present in the lake. To the best of 
our knowledge, this study represents the first detailed 
characterization of Chironomidae in the Lake Victoria region 
of Kenya. The currently available data and reports tend to 
group all the available species into lake flies. Sequencing 
of the partial genes ascertained the high degree of genetic 
divergence of Chironomidae in the lake region, which is a 
starting point for further research. Although we recorded 7 
species of Chironomidae in the Lake Victoria ecosystem of 
Kenya for the first time, the species richness of Chironomidae 
is likely much higher due to the small sample sizes collected 
in our study. Although for some genera such as Chironomus 
and Polypedilum, molecular approaches are very important, 
they do not always solve taxonomic problems. 
Chironomidae are not considered a nuisance by the 
rural communities along the shores of Lake Victoria in 
Kenya. Rather, these insects are considered important 
sources of protein for the rural local communities residing 
in the Lake Victoria region of Kenya. Therefore, it necessary 
to study the nutritional profile of all species prevalent in 
the region. Future studies should also establish the relation-
ship between diversity of the Chironomidae with respect to 
the environmental variables of Lake Victoria and its sur-
roundings so as to design effective conservation strategies 
that target the insects. Several studies have linked corre-
lation of environmental variables were with the composi-
tion of chironomid communities (Belle and Goedkoop, 2021, 
Leszczyńska et al., 2021, Čerba et al., 2022). Climate change 
induced changes in precipitation patterns and tempera-
tures are affecting the ecosystem of the Lake Victoria region 
(Akurut et al., 2014, Luhunga and Songoro, 2020, Ototo et al., 
2022) which in turn can have potentially profound effects 
on the diversity and distribution of the chironomids. In 
addition, landscape inputs from urban and/ or agricultural 
areas are probably also important in influencing the diver-
sity and distribution of the chironomids. For example, the 
locals harvesting these insects for food have reported less 
sightings of the chironomids, and this is likely attributable 
to the changing climate and other factors such as human 
activities. Thus, it is necessary to monitor the population 
dynamics of the chironomids over a longer period of time 
to further enhance our understanding of the long-term 
impacts of climate change on the diversity and distribu-
tion of these edible insects. As a key component to future 
monitoring activities, a peer reviewed reference collection 
of the specimens collected in this study is established, and 
will be used to confirm populations over time and across 
different localities. Moreover, cleared specimens of adults 
(not just wings) will need to be permanently slide mount to 
create a reference collection that will be housed at an insect 
museum to help establish a good collection with long term 
curation for future research and will benefit researchers in 
the long run. Also, future studies should focus on the ecolog-
ical impacts of chironomids in the Lake Victoria region by 
investigating how subsidies of the chironomids affect litter 
processing and microbial communities, and evaluating how 
those belowground effects related to changes in inorganic 
nitrogen, plant composition and net primary productivity.

Diversity Analysis of Adult Chironomidae in the Lake Victoria Basin of Kenya
51
Acknowledgements 
This study was funded by the Africa Center of Excellence in 
Sustainable Use of Insects as Food and Feeds (INSEFOODS) 
of Jaramogi Oginga Odinga University of Science and 
Technology, and the World Bank.
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Analiza raznolikosti odraslih trzač (Chironomidae) v 
obalnem pasu Viktorijinega jezera v Keniji
IZVLEČEK
Trzače (Chironomidae) naseljujejo večino vodnih habitatov na Zemlji in pogosto prevladujejo v združbah vodnih žuželk, tako 
v številčnosti kot tudi bogastvu vrst. Kljub njihovemu ekološkemu pomenu pa diverziteta in porazdelitev trzač v ekosistemu 
Viktorijinega jezera v Keniji do danes nista bili raziskani. V članku poročamo o diverziteti in porazdelitvi odraslih trzač na obalnih 
območjih Usenge, Mbita in Ogal ob Viktorijinem jezeru, z uporabo morfoloških značilnosti, kot tudi na osnovi sekvencioniranja 
gena, ki kodira citokrom c oksidazo, podenoto 1 (COI). Z mikroskopsko karakterizacijo na podlagi venacije kril smo identificirali 
štiri rodove, Tanypus, Coelotanypus, Dicrotendipes in Chironomus. Črtno kodiranje gena COI je nadalje razkrilo več rodov/vrst, 
vključno s Kiefferulus brevibucca, Chironomus flaviplumus, Polypedilum fuscovittatum, Polypedilum sp. in Dicrotendipes sp. 
Identificirane vrste so bile razvrščene v tri skupine s pomočjo filogenetske metode 'povezovanja sosedov'. Med tremi preučevanimi 
območji smo opazili razlike v bogastvu vrst, pri čemer je bilo na obali Mbita ugotovljeno največje bogastvo vrst. Evolucijska analiza 
je razkrila sorodnost med vsemi identificiranimi vrstami, kar kaže na nedavnega skupnega prednika. Raziskava predstavlja prvo 
poročilo o podrobni karakterizaciji trzač (Chironomidae) v ekosistemu Viktorijinega jezera v Keniji. Raziskava poleg tega služi kot 
prvi korak k celovitemu razumevanju predstavnikov vrst iz družine Chironomidae, ki naseljuje ta ekosistem. 
Ključne besede: Chironomidae, citokrom c-oksidaza podenota 1, črtno kodiranje DNA, venacija kril, raznolikost