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Comprehensive Research Journal of Biological Science (CRJBS) Vol. 2(2) pp. 034 - 042 July, 2014
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Full Length Research Paper
The Blackfly Species (Diptera: Simuliidae) On the
Campus of the Obafemi Awolowo University and Their
Polytene Chromosomes
Adewale A. Sorungbe
Department of Biology, Federal University of Technology, P. M. B. 704 Akure, Ondo State, Nigeria.
E-mail: [email protected]
Accepted 25 July, 2014
The polytene chromosomes of four blackfly species found on the campus of the Obafemi Awolowo
University along with some diagnostic features of the larval and pupal stages were studied using
standard techniques. The four species were Simulium alcocki Pomeroy, Simulium damnosum Theobald
sensu lacto and two unidentified species of Simulium herein referred to as US 1 (unidentified species 1)
and US 2 (unidentified species 2). The results were obtained using the salivary glands from penultimate
to final larval instars. Polytene chromosomes were observed in all species. There is no chromocentre,
and all the three chromosomes are discrete in the four species studied. Features such as puffs, short
sections of asynapsis and inversions could be identified. The salivary gland polytene chromosomes of
Simulium species 2 (US 2) were extremely fragile and hence proved difficult to handle. From the results
of this study, one can concluded that blackfly species may be conclusively identified using the larval
and pupal morphological characteristics as well as their polytene chromosome characteristics.
Keywords: Simulium alcocki, Simulium damnosum, sensu lacto, polytene, chromosomes.
INTRODUCTION
Blackflies are small stout bodied insects; body colour
varies widely as not all blackflies are black. They are
cosmopolitan
in
distribution
(Crosskey,
1990).
Subdivision of the genus Simulium in the early days of
simuliid taxonomy was based on the following; structure
of the second hind tarsal segment, wing venation in the
imago, structure of the cocoon and the number of
respiratory filaments in the pupa (Crosskey, 1990). The
blackfly is the vector of the disease onchocerciasis
caused by the filarial nematode Onchocerca volvulus
Leuckart (Nematoda: Filarioidea), with about 17.8 million
people affected worldwide. Onchocerciasis is a very
debilitating disease with heavy infection resulting in
blindness and intense puritus (Wilson and Post, 1994;
Waddy, 1969). A skin condition known by the appellation
craw-craw (pidgin English) recognized around the leg
area and accompanied by intense itching is another
manifestation of onchocerciasis in the forest region.
(Waddy, 1969).
The blackfly is unique in a number of ways amongst
which are that it is composed of about 1,554 species,
described and named (morphological and chromosomal),
both larval and pupal stages possess useful taxonomic
characters for identifying species such as the larval
respiratory histoblast and the shapes and sizes of pupal
respiratory gills, a high rate of sibling speciation within the
Sorungbe 035
genus, with morphological look alike which are
biologically separate species in nature (Freeman and de
Meillion 1953; Crosskey 1990).
Cytological evidence has shown that they have an
extraordinarily high rate of sibling speciation. Similar
characteristics have been reported in other insects such
as the mosquito genera Anopheles (Davidson, 1974),
Culex, and many of the fruitflies of the family Tephritidae
(Berlocher, 1978).
Polytene chromosomes are known by many names
such as giant chromosomes, megachromosomes, etc.
they are giant chromosomes occurring in the salivary
glands and other tissues of many Diptera, including the
blackfly Simulium.
In the Simuliidae, analysis of polytene chromosome
salivary chromosomes of larvae has contributed
significantly to evolutionary and cytotaxonomic research.
Additionally, such studies have had practical value in
efforts to control onchocerciasis.
Knowledge of the cytology of blackflies found in South
Western Nigeria in general is extremely scanty and
especially the more in the Ile-Ife area, coupled with the
fact that cytology is a useful tool in the identification of
blackfly species and with the need to provide valuable
information necessary for the control of the
dermatological manifestation of the disease; a form
common in South Western Nigeria. In West Africa, it is
transmitted exclusively by members of the Simulium
(Edwardsellum) damnosum. Theobald complex (Diptera:
Simuliidae). The sibling species within this complex have
been distinguished by chromosomal (cytotaxonomic)
features, and their accurate identification remains difficult.
MATERIALS AND METHODS
The campus of the Obafemi Awolowo University Ife-Ife,
0
1
Nigeria is situated approximately between Latitude 7 27
0
1
0 1
0
1
and 7 35 North and Longitude 4 3 and 4 39 East.
Simulium larvae were collected at four sites (two
perennial streams and two rivers). These water bodies
were selected as collecting sites based on the following
criteria (i) the presence on these water bodies of
sections, which are fast flowing, (ii) abundance of blackfly
Simulium larvae and pupae (Fig 1).
Larvae were collected at the various sites early in the
morning using flexible forceps and were fixed
immediately in freshly prepared 1:3 Glacial acetic acid
Ethanol (Clarke’s Fluid) for 3-4 hours which had been
previously cooled in a fridge for at least one hour before
use. Pupal stages were also collected simultaneously
and used to positively identify larval stages since
species- specific characters used for positive
identification of blackfly species exist both in the larval
and pupal stages. Some pupae of the various species
were reared to adulthood and preserved as demanded by
standard simulid taxonomic procedures. In the laboratory,
the abdomen of the larvae was cut open to permit
exposure of the salivary glands to the action of the rapidly
penetrating Clarke’s fluid as proposed by Dunbar (1972).
Identification was carried out with the aid of a
dissecting microscope using the diagnostic larval
respiratory filament or histoblast and the pupal respiratory
gills of pupae collected at the same time as the larval
stages. Confirmation was done using the key produced
by Freeman and de Meillon (1953).
Dissection was carried out in 5% hydrochloric acid by
totally cutting open the body wall and exposing the paired
salivary glands. The salivary glands were left in the
hydrochloric acid for between 3-5minutes and then
stained in a drop of FLP orcein (Olorode, 1974) for 15
minutes.
A cover slip was applied and squashing was carried out
with the thumb. The slide was then placed in between a
fold of filter paper and pressure gently applied to remove
excess stain. Prepared slides were scanned at high and
low magnifications of a compound light microscope and
photomicrographs were taken.
Nomenclature
chromosome
of
salivary
gland
polytene
Cytotaxonomists describe the position of chromosomal
features in Simuliidae by a reference convention in which
the paired double ‘chromosomes’ are numbered I-III in
decreasing order of length and their arms (parts lying on
either side of the centromere region) using Roman
numerals. Long and short arms are indicated by the
capital letters L or S written after the chromosome
number (IS, IIIL, etc). Morphological characters are
denoted using the first letter of their names in capital
letter such as centromere C, puff P, inversion I, etc
(Bedo, 1977, Crosskey, 1990).
Results
In all, four species of the blackfly Simulium were found on
the campus of the Obafemi Awolowo University, Ile Ife.
These are Simulium alcocki Pomeroy, Simulium
damnosum Theobald and two unidentified species herein
referred to as US 1 (Unidentified species 1) and US 2
(Unidentified species 2).
Simulium damnosum Theobald s.s. and US 2 are the
least abundant as they can be found at a single site each
while US one can be found at three of the four sites
sampled within the university campus. A noteworthy
observation is that larvae of different species found at a
site will be seen on the same submerged leaf, stick, or
stone, same for the pupae. Very rarely were three
species found on the same submerged object or at the
036 Compr. Res. J. Biol. Sci.
Table 1. Distribution of the various species of the blackfly Simulium at four sites on the campus of the
Obafemi Awolowo University, Ille-Ife.
Fig. 2: Diagram of Simulium alcocki Pomeroy showing;
(a)
Larva with larval respiratory histoblast
(b)
Pupa with pupal respiratory filaments showing diagnostic 2-3-2 arrangement (Top view)
(c)
Side view of pupa
Legend:
l.r.h.: larval respiratory histoblast
p.r.f.: pupal respiratory filament
c: cocoon
same site.
Morphological Characteristics
Simulium alcocki Pomeroy
Shape: elongate, with usual club shaped mid-section
which enlarges gradually towards the posterior end.
Colour: light brown, some older larval instars have head
pigmentation similar to that of S. damnosum
Shape of histoblast (larval respiratory organ): consist
of a bundle of tightly coiled histoblast in form of a tube
with an open end, pointing towards the dorsal side of the
body. It is made up of six to nine turns which in most
cases cannot be differentiated from one another and in
some cases, a small clear central areas exists in the
histoblast.
Setae: present but not conspicuous
Prolegs: long, curve (in some cases), thick and pointed
Length: 3mm to 5.5mm
Simulium damnosum Theobald
Shape: almost barrel shaped, swollen near the beginning
of the mid-section immediately after the head. Reduced
in size around the mid section and increases towards the
Sorungbe 037
Fig. 3: Diagram of Simulium damnosum Theobald showing;
(a) Larva with larval respiratory histoblast
(b) Pupa with pupal respiratory filaments showing diagnostic (Top view)
(c) Side view of pupa
Legend:
l.r.h.: larval respiratory histoblast
p.r.f.: pupal respiratory filament
c: cocoon
Fig. 4: Diagram of Simulium species (US1) showing;
(a)
Larva with larval respiratory histoblast
(b)
Pupa with papal respiratory filaments showing diagnostic (Top view)
(c)
Side view of pupa
(d)
Side view of pupa
Legend:
l.r.h.: larval respiratory histoblast
p.r.f.: pupal respiratory filament
c: cocoon
swollen end. Possesses distinctive conical (mole hill
shaped) dorso- lateral tubercles (all with tiny setae/hairs
on most of the thoracic segments).
Colour: the head is distinctively dark brown with the
darkest area around the eye spots. The body is greyish
and some are dark grey all over.
Shape of histoblast (larval respiratory organ): The
shape is distinctive for S. damnosum; unmistakable in
that it resembles a rosette with a large central area.
Setae: present and conspicuously visible on every body
segment as small conical tuft on the dorsal cuticle and on
the general larva body giving the larva a hairy and dark
appearance.
Prolegs: short, conical and pointed.
Length: 3mm to 5.5mm
US 1 (Unidentified species 1)
Shape: the mud-section is swollen, becomes narrow
038 Compr. Res. J. Biol. Sci.
Fig. 5: Diagram of Simulium species (US2) showing;
(a)
Larva with larval respiratory histoblast
(b)
Pupa with pupal respiratory filaments showing diagnostic (Top view)
(c)
Side view of pupa
Legend:
l.r.h.: larval respiratory histoblast
p.r.f.: pupal respiratory filament
c: cocoon
Fig. 6 & 7: FLP orcein stained salivary gland polytene chromosomes of Simulium
A.
Complement of S. alcocki Pomeroy showing gross features
B.
Complements of S. damnosum Theobald showing gross features
Legend: AR - Asynaptic region
P - Puff
I - Inversion
C - Centromere
towards the middle, with the abdomen being club shaped
and elongate.
Colour: the head is creamy, almost whitish, edges of the
head capsule darkened (dark brown). The body is
brownish in colour, with some creamy/yellowish areas.
Shape of histoblast (larval respiratory organs):
resembles a leaf with lines that look like veins on the
surface of a leaf and running to the edges. Resembles
two leaves fused into one
Setae: conspicuous especially on the dorsal side of the
abdomen sparse in distribution.
Prolegs: Thin, long and pointed
Length: From 3.5mm to 5.5mm
Sorungbe 039
Fig. 8: FLP orcein stained salivary gland olytene chromosomes of Simulium species
A.
Chromosome I of Simulium species (US 1)
B.
Chromosome II of Simulium species (US 1)
Note asynapsis at two points and centromere C
C.
Chromosomes III of Simulium species (US 1)
Legend: AR – Asynaptic region
C - Centromere
US 2 (Unidentified Species 2)
Shape: elongate like S. alcocki. Mid-section enlarges
gradually towards the posterior end.
Colour: light brown in colour, head capsule pigmented
especially around the eye spots (brown) with a clear zone
in the immediate area around the eyespots giving the
larva the image of having a ‘black-eye’.
Shape of histoblast (larval respiratory organ):
resembles that of S. alcocki. The shape is disfigured and
cross striations breaking it into sections. There is
variation in this basic structure in younger larval instars.
Setae: not conspicuous
Prolegs: distinctively short and stout
Length: 3mm to 6mm
Salivary gland polytene chromosomes
The best spreads were obtained from freshly collected
samples in which the abdomen was cut open and fixed
for between three to five hours before dissection.
A rigorous comparison within the possible limits
permitted by direct microscopic and photomicrographic
observations reveals identical banding patterns within
each species. Puffs were observed in most spreads.
Local asynapsis was a common feature observed in all
species under investigation. This is observed as a
separation of the homologous chromosomes over a short
length of the chromosome, as seen in Fig. 6 and fig. 7.
Simulium alcocki Pomeroy
Salivary gland polytene chromosome strands follow the
normal complement of three single strands. The arms of
this species were long and thin. Asynapsis was observed
in short segments along the length of the chromosomes
of most slides. In fig. 6, puffs can be observed on the
short arm (IIIS) of chromosome III and on the short arm
(IIS) of chromosome II. A small inversion was also
observed on the short arm (IIIS) of chromosome III, a few
bands from the large puff on the same arm i.e. IIIS.
Landmark features like nucleolar organizer, Balbiani
ring and parabalbiani were absent.
Though puffs were observed in most spreads of all
species of blackfly studied, no definite statement can be
made concerning the specificity or regularity of such
040 Compr. Res. J. Biol. Sci.
Fig. 9: FLP orcein stained salivary gland olytene chromosomes of Simulium species
(A)
Chromosome I of Simulium species (US 1)
(B)
FLP orcein stained salivery gland polytene Chromosomes complement of Simulium
species (US 2)
Legend: C - Centromere
IL - Long Arm of Chromosome I
IS – Short Arm of Chromosome I
Fig. 10: Enlarge photomicrograph of long arm chromosome I Simulium species (US 2).
Legend: P – Puff.
bands, as there was no way of obtaining blackfly larvae
which are all at the same stage of development.
Simulium damnosum Theobald s.s.
The normal salivary
gland
polytene
chromosome
Sorungbe 041
complement was observed in this species. In terms of
width, the arms of S. damnosum are wider. s
Regions of asynapsis were also observed in most
spreads of this species. As seen in Fig. 6B, puffs could
be observed on both chromosomes II and III. Two large
inversions denoted I1 and I2 can be identified on
chromosome II. Due to the inability to identify the
centromere, their precise location on either the short or
long arm of chromosome II cannot be stated. Features
resembling landmarks such as nucleolar organizer,
balbiani and parabalbiani could not be positively
identified.
Simulium species (US I)
The normal salivary gland polytene chromosome
complement was observed. Regions of asynapsis could
also be observed in this species as with others as well as
the centromere.
the spreads was the asynapsis of homologues, which
occurred along short lengths of the larval silk gland
polytene chromosomes.
A heavy orcein-staining band across a prominent
expanded region in each chromosome marks the
centromere, unlike Drosophila where the centromere of
all the chromosome fuse to form the chromocentre, this is
not the case in Simulium, the only exception in this genus
is in an unnamed species (White, 1979). Some very large
specialized puffs are referred to as Balbiani rings (White,
1979) while the parabalbiani, a large darkly stained band
with one sharply defined and one diffuse edge, serves as
useful markers. The parabalbiani is unique to the
Simuliidae (Bedo, 1977) but could not be positively
identified in this study.
From the result of these studies carried out on these
four species of blackfly, centromeric position, shape of
larval respiratory histoblast, pupal respiratory filament
structure
and
landmark
features
of
polytene
chromosomes were among the reliable induces which
were used to positively identify blackfly species at least to
the generic level.
Simulium species (US 2)
Salivary gland polytene chromosomes of this particular
species proved extremely difficult to handle. As with other
species, the normal chromosome complement was
observed as with regions of asynapsis, centromere and
puffs.
DISCUSSION AND CONCLUSION
In the Simuliidae, analysis of polytene chromosome
salivary gland chromosomes of larvae has contributed
significantly to evolutionary and cytotaxonomic research.
Additionally, such studies have had practical value in
efforts to control onchocerciasis (Bedo, 1976).
All the species of blackfly investigated in this study
share the same basic three chromosomes of the family
Simuliidae. This is in agreement with the results obtained
by other workers such as Dunbar (1969), Vajime and
Dunbar (1975), Mafuyai, et al (1996).
Puffing patterns in all species studied differ but were
relatively constant within the species. This is supported
by the nature of puffs as reported by other workers
especially Drosophila, Chironomus, etc in that they
appear and regress based on the developmental stage
attained by a given species in a given chromosome
(Ashburner, 1969). The puffs are believed to be sites of
active ribonucleic acid (RNA) synthesis and therefore a
mechanism for the amplification of localized regions of
the genome.
Polymorphic inversions commonly found in the larval
population of blackflies with sibling species were
recognized in this particular study, one in S. alcocki and
two in S. damnosum. A common observation in most of
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