The Hypopharynx of Male and Female Mosquitoes

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The Open Entomology Journal, 2007, 1, 1-6 1
1874-4079/07 2007 Bentham Science Publishers Ltd.
The Hypopharynx of Male and Female Mosquitoes
§
Isra Wahid
*,1
, Toshihiko Sunahara
2
and Motoyoshi Mogi
2
1
Department of Parasitology, Hasanuddin University School of Medicine, Jl. Perintis Kemerdekaan KM. 11 Tamalanrea,
Makassar 90245, Indonesia
2
Department of Parasitology, Saga University School of Medicine, Nabeshima 5-1-1, Saga Shi, Saga Ken, Japan
Abstract: In blood-feeder female mosquitoes, the hypopharynx stylet is one part of the fascicle, the structure that pierces
into the host skin during blood feeding. As other parts, the hypopharynx is a free stylet. However, since male mosquitoes
do not feed blood, their mouthparts are less developed. The hypopharynx fuses with the inner wall of the labium, while
maxillae and mandibles are much shorter than the labium. Only the labrum and the labium are well developed and func-
tion as food canal and its sheath, respectively. Light microscopy (LM) and scanning electron microscopy (SEM) were
done to compare the hypopharynx of males of several mosquito genera and, in addition, females of autogenous mosqui-
toes.
The hypopharynxs of males of both autogenous and anautogenous mosquitoes fuse with the labium inner wall as long as
the labium length, but are distinctly different structures from the labium. Dissociation occurs on the hypopharynx of fe-
male autogenous mosquitoes: Toxorhynchites spp. have a free hypopharynx as in anautogeny mosquitoes, whereas it fuses
with the labium wall in Malaya genurostris Leicester, as in male mosquitoes.
INTRODUCTION
As most of female mosquitoes feed on blood, their
mouthparts are highly specialized for piercing the host skin
and sucking blood. During blood feeding, the fascicle (con-
tains a labrum, a hypopharynx, pairs of mandibles and max-
illae) enters the host skin, while the labium-formed sheath
remains outside the skin. The maxilla and the mandible func-
tion as piercing organs [1,2], but there is no active movement
of the hypopharynx. No muscles are either originated from
or inserted on the hypopharynx [2]. Thus, practically, there is
no active movement of the hypopharynx. Its function is to
transfers saliva to the host tissue through salivary canal on its
dorsal surface.
Unlike females, male mosquitoes do not feed on blood.
Vizzi described the hypopharynx in Anopheles quadrimacu-
latus Say males as a sclerotic plate on the labial gutter [3],
while Snodgrass stated that the hypopharynx and the labium
of male mosquitoes completely fuse together forming the
labio-hypopharynx [4]. Christophers recognized the hypo-
pharynx of Aedes aegypti (Linneaus) males as a ridge on the
labium [5]. Though the hypopharynx of Ae. aegypti males is
not free from the labium wall, it is discernible from the la-
bium wall by its texture both under light and scanning elec-
tron microscopy (SEM). Fusion of the hypopharynx with the
labium is understandable, because the hypopharynx of males
does not need to enter into the skin.
*Address correspondence to this author at the Department of Parasitology,
Hasanuddin University School of Medicine, Jl. Perintis Kemerdekaan KM.
11 Tamalanrea, Makassar 90245, Indonesia; Tel: +62-411-580687; Fax:
+62-411-586297; E-mail: israwahid@gmail.com
§
Note: Some parts of this work were presented in The 2
nd
ASEAN Congress
of Tropical Medicine and Parasitology, Bandung, Indonesia, May 21-23,
2006).
MATERIALS AND METHODS
Mosquitoes
Materials used were the same with those used for exami-
nation maxillae and mandibles of male mosquitoes in our
previous study [1] listed in Table 1. The hypopharynx states
of males of 44 species of 12 genera, females of 2 partially
autogenous and 5 autogenous species were examined. For
comparison, the hypopharynx of female Ae. aegypti was
examined as a representative of anautogenous species.
Specimens were kept in 70% ethanol solution, except for a
few specimens kept in 3% glutaraldehide solution for trans-
mission electron microscopy (TEM).
Light Microscopy
Males of all the 44 species were examined under light
microscopy. The specimens were stained with Fuschin Acid
as desribed in Wahid et al. [6]. They then put in methyl cel-
losolve (Nakarai Chemicals, Kyoto, Japan) on a slide glass
and dissected under a dissecting microscope. The head was
separated from the thorax. The free hypopharynx of females,
together with other free stylets, was easily pried up from the
labium gutter by a fine insect pin. On the other hand, it was
no easy to separate the hypopharynx of males from labium
tissue. The clypeus was pulled upward and forward care-
fully, to separate the hypopharynx from the inner wall of the
labium. The detached hypopharynx was then cover with
cover glass and examined under a light microscopy.
Scanning Electron Microscopy (SEM)
SEM was used to confirm the observation made under
light microscopy. For this purpose, only males of Ae. aegypti
were examined, since all male specimens principally have
the same attribute. The dissected head, taking the labrum,
maxillae and mandibles out of the labial gutter and leaving
the hypopharynx undisturbed, was put on a poly L lysine-
coated glass slide. The specimens were gradually dehydrated

2 The Open Entomology Journal, 2007, Volume 1 Wahid et al.
through 70% to 99% ethanol, and then placed in 99% t-butyl
alcohol and kept in a freezer (-20
o
C). They were freeze-dried
by using an ID-2 drier (EIKO, Tokyo, Japan) prior to gold
coating by an EIKO IB-3 ion coater, then examined by a
JSM-5200 LV scanning electron microscope (JEOL, Tokyo,
Japan).
Transmission Electron Microscopy (TEM)
Transversal section of the proboscis of males and females
of Ae. aegypti, Malaya genurostris Leicester, Topomyia
vijayae Ramalingan and Toxorhynchites splendes (Weide-
mann) were examined by TEM. Fresh or dry specimens were
kept in 3% glutaraldehyde solution (dry specimens were first
put in 70% ethanol for ± 5 seconds to make them sinkable
completely into 3% glutaraldehyde). Heads were then sepa-
rated from the rest of the body in 3% glutaraldehyde solution
buffered to pH 7.4 with phosphate buffer, then fixed for at
least 1 hr in phosphate-buffered 1% osmium tetroxide solu-
tion. Dehydration was carried out by passing the head
through 50%, 75%, 85%, 95%, 100% ethanol, each for 15
min, then, head was kept in n-glycildiethyl solution (QY-1)
for 20-40 min, to a 1: 1 mixture of Epon and QY-1 for 1 hr,
in a 3: 1 mixture of Epon-QY-1 overnight, and was embed-
ded in Epon 812 for 2 h before heating at 45

C and 60

C,
each for 24 h. The block was then cut with an ultra micro-
tome. Sections were mounted on parlodion-carbon coated
grids and stained in a saturated uranyl acetate solution fol-
lowed by lead citrate. Specimens were examined by a trans-
mission electron microscope JEM-1210 (JEOL, Tokyo, Ja-
pan).
RESULTS
Hypopharynx of Males
The hypopharynx of male mosquitoes invariably fuses
with the inner wall of the labium for all the 44 species (Table
1). It is a thick longitudinal plate ventrally fused with the
inner wall of the labium. Its proximal and distal ends connect
to the cibarial ventral wall and the hairy ligula at the probos-
cis tip, respectively (Fig. 1). Contrasting to the longitudinally
wrinkle surface of inner wall of the labium, the hypopharynx
has a smooth surface and a gutter-like salivary canal at the
median line (Fig. 2).
By light microscopy, the hypopharynx of males can be
recognized as a longitudinal structure in the labial gutter
with parallel edges and a midrib representing a salivary ca-
nal. A hypopharynx that is detached from the labium has two
parallel edges at both sides. The inner edge is the hypophar-
ynx edge and the outer one is the labium wall tissue that is
attached to the hypopharynx (Fig. 3). The transversal section
of the proboscis by transmission electron microscopy (TEM)
shows different electron density between the hypopharynx
tissue and the labium tissue (Fig. 4).
Anatomically, the salivary canal is a dorsally open gutter
on the hypopharynx surface. However, its overlapping edges
make it functionally a closed tube (Fig. 4A-C), distally
opened at the tip of hypopharynx (Fig. 1).
Hypopharinx of Females
The hypopharynx of female mosquitoes almost invaria-
bly are separated from the labrum as a free stylet. Females of
Fig. (1). The hypopharynx tip Ae. aegypti males fused with the
ligula. HP, Hypopharynx. SC, Salivary canal. LG, Ligula.
Fig. (2). The hypopharynx trunk of Ae aegypti male inside the la-
bium gutter. HP, Hypopharynx. HE, The hypopharynx edge. SC,
Salivary canal. IW, Inner wall of labium. OW, Outer wall of la-
bium.

The Hypopharynx of Mosquitoes The Open Entomology Journal, 2007, Volume 1 3
anautogenous mosquitoes, represented by the well known
domestic mosquitoes Ae. aegypti is a stylet that completely
separated from labrum as shown in TEM image (Fig. 5).
Mosquitoes that only autogenous at the first batch of their
eggs, but ultimately need blood feeding for the rest of their
eggs, as predicted, have hypopharynx attributes similar to
those of anautogenous one. The females of Culex pipiens
molestus Forskal and Ochlerotatus togoi (Theobald), are
examples of this group, the partially autogenous species.
However, despite the distinct non blood-feeding habits of
females Toxorhynchites spp. and Topomyia spp., these life-
time autogenous mosquitoes showing hypopharynx charac-
teristics similar to those of the autogenous females, a free
stylet hypopharynx (Fig. 6A,B). The attributes contrasting to
those of another lifetime autogenous species, Malaya
genurostris, which females have a male like-fused hypo-
pharynx (Fig. 7).
Fig. (3). The detached hypopharynx of a male Ae. aegypti from the
labium under a light microscope showing a double edges at each
side. The inner edge is the hypopharynx edge (HE), while the outer
edge is the attached labium tissue (LT). SC, Salivary canal.
Table 1. Status of Hypopharynx of 44 Species Examined
Species Female Male
Aedes aegypti (Linneaus) Free Fused
Aedes albopictus (Skuse) Free Fused
Aedes flavopictus Yamada Free Fused
Aedes galloisi Yamada Free Fused
Aedes paullusi Stone and Farner Free Fused
Aedes pseudoalbolineatus Brug Free Fused
Aedes riversi Bohart and Ingram Free Fused
Aedes scutellaris (Walker) Free Fused
Anopheles kochi Doenitz Free Fused
Anopheles lindesayi japonicus Yamada Free Fused
Anopheles sinensis weidemann Free Fused
Anopheles stephensi Liston Free Fused
Anopheles vagus Doenitz Free Fused
Armigeres sp. 1 Free Fused
Armigeres sp. 2 Free Fused
Armigeres sp. 3 Free Fused
Armigeres subalbatus (Coquillett) Free Fused
Armigeres theobaldi Barraud Free Fused
Culex fuscanus weidemann Free Fused
Culex halifaxii Theobald Free Fused
Culex kyotoensis Yamaguti and La Casse Free Fused
Culex pipiens molestus Forskal
a
Free Fused
Culex pipiens pallens Coquillett Free Fused
Culex quinquefasciatus Say Free Fused
Culex tritaeniorhyncus Giles Free Fused
Malaya genurostris Leicester
b
Fused Fused
Mimomyia chamberlaini metallica (Leicester) Free Fused
Fig. (4). Transversal sections of the hypopharynx of males of Ae aegypti (A), Tx splendens (B), and To. vijayae (C). FC, Food canal of the
labrum. HP, Hypopharynx. LB, Labium. SC, Salivary canal.

4 The Open Entomology Journal, 2007, Volume 1 Wahid et al.
(Table 1) contd…..
Species Female Male
Ochlerotatus japonicus japonicus (Theobald) Free Fused
Ochlerotatus poecilius (Theobald) Free Fused
Ochlerotatus togoi (Theobald)
a
Free Fused
Orthopodomyia anopheloides (Giles) Free Fused
Orthopodomyia sp. Free Fused
Topomyia vijayae ramalingan
b
Free Fused
Topomyia yanbarensis
b
Free Fused
Tripteroides aranoides (Theobbald) Free Fused
Tripteroides bambusa bambusa Yamada Free Fused
Tripteroides sp. 1 Free Fused
Tripteroides sp. 2 Free Fused
Toxorhynchites amboinensis (Doleschall)
b
Free Fused
Toxorhynchites manicatus yaeyama Bohart
b
Free Fused
Toxorhynchites splendens (weidemann)
b
Free Fused
Uranotaenia novobscura novobscura Barraud Free Fused
Uranotaenia sp. 1 Free Fused
Uranotaenia sp. 2 Free Fused
a
Partially autogenous species;
b
Lifetime autogenous species.
DISCUSSION
The present study confirmed the previous reports, that
hypopharynx of male mosquitoes fuse with the labium,
forms the labio-hypopharynx [4]. Although the tissue of the
hypopharynx part in the labio-hypopharynx is clearly differ-
ent from the tissue of the labium part. Males of all the 44
species representing 12 mosquito genera invariably have a
hypopharynx fused with inner wall of the labium, regardless
feeding habits of their females. Thus, this attribute was es-
tablished probably at the earliest stage in mosquito evolu-
tion, before extensive diversification.
Fig. (5). Transversal section of the proboscis of an Ae. aegypti fe-
male. The free hypopharynx (HP) is separated from the labium
(LB) by a space accommodating the maxillae (MX). FC, Food ca-
nal. LR, Labrum. MD, Mandibles. SC, Salivary canal.
It is interesting to note that males of a sand fly, Lutzo-
myia migoney Franca (Psychodidae) has a free hypopharynx
[7] and males of black flies of the genus Simulium (Simuli-
dae) has a free hypopharynx trunk but its tip fuses with the
ligula by a thin transparent membrane (personal observation,
unpublished data). Fusion of the hypopharynx and the la-
bium may have taken place after separation of the psychodid
line from the lineage including the ancestors of Simulidae
and Culicidae.
Despite its fusion with the labium wall, the hypopharynx
reaches the tip of the proboscis, with the salivary canal on its
Fig. (6). Transversal sections of the proboscis of autogenous females of Tx. splendens (A), To. vijayae (B) showing the free hypopharynx.
FC, Food canal. HP, Hypopharynx. LB, Labium. LR, Labrum. SC, Salivary canal.

The Hypopharynx of Mosquitoes The Open Entomology Journal, 2007, Volume 1 5
dorsal surface. Differing from male mandibles and maxillae
that probably have no function, the salivary canal of males
functions as a closed tube by its dorsal overlapping edges, as
in females [1,2,8].
Fig. (7). Transversal section of the proboscis of a Ml. genurostris
female showing its unique hypopharynx fused with the labium as in
males. FC, Food canal. HP, Hypopharynx. LB, Labium. SC, Sali-
vary canal.
Saliva secreted by salivary glands of males contains -
glucosidase, -amylase and bacteriolytic factors, but lack the
polypeptide D7, sialokinins I and II, and apyrase which are
secreted from the median lobe and the distal portion of lat-
eral lobes of female’s salivary glands [2]. The latter three
enzymes are responsible for immunoreaction of hosts [9],
endothelium-dependent vasodilatation [10] and anti blood-
coagulation [11-13], respectively. Apparently males need
saliva for digestion of their main food such as floral and ex-
tra-floral nectar and honeydew [14], as well as for protection
from pathogenic bacteria in sugar sources [15].
The present study confirmed the previous reports for the
free hypopharynx of anautogenous females [4, 5, 8,16-23].
For autogenous females, Hudson reported that an autogenous
population of Wyeomyia smithii has a free hypopharynx [20].
We described for the first time, the hypopharynx of fe-
males of two partially autogenous (Cx. pipiens molestus and
Oc. togoi) and six lifetime autogenous females (3 species of
Toxorhynchites, 2 Topomyia and 1 Malaya). As in anautoge-
nous mosquitoes, females of life-time autogenous Toxorhyn-
chites and Topomyia, as well as partially autogenous Cx.
pipiens molestus and Oc. togoi have a free hypopharynx.
However, females of autogenous Ml. genurostris have
shown a fused hypopharynx with the labium wall as the hy-
popharynx of males.
The unique case of Ml. genurostris, previously known as
Harpagomyia [24, 25], may correlate with its remarkable
feeding habit among mosquitoes that they are acquiring food
from a regurgitation fluid by Crematogaster ants [25-30]
with its peculiar proboscis that swollen at the tip [25]. Jacob-
son [26,27] in Clements [30] described that adults of Malaya
splendens feed on a black tree ant, Crematogaster difformis.
The adult mosquitoes positioned them self on the ant trails,
head uppermost, and rocked back and forth, left and right.
Ants ascending the tree walked unhindered between the legs
of the mosquitoes, but when an ant descending the trunk
reached a mosquito, the mosquito palpated the ant’s head
with its forelegs and antennae. Usually the ant stopped,
pressed its thorax to the tree while raising its abdomen and
opening its jaws widely. The mosquito immediately started
rocking forward and backward while vibrating its wings.
When the ant regurgitated a drop of liquid, the mosquito
imbibed it, after which the ant continued on its way.
There are two possibilities of the evolution of hypophar-
ynx of genus Malaya: Firstly, members of the genus retained
an old attribute structure of mosquito ancestor’s hypophar-
ynx that might be fused before its separation from labium for
the purpose of sucking blood. However, this hypothesis is
unlikely since a free hypopharynx, as in anautogenous spe-
cies, is the most common state in female mosquitoes that
suggest that their common ancestor had this attribute [31].
More far, the old genera Anopheles that is distributed world
wide and placed on the basis of phylogeny tree of mosqui-
toes based on clasditic analysis [32] has share the free hypo-
pharynx attributes, as well as other autogenous mosquitoes
mention above, and hence showing that the hypopharynx of
female mosquitoes has a free origin from their common an-
cestor.
The second hypothesis regarding the attribute of female
Malaya hypopharynx is that the fused hypopharynx of this
genus might recently derived from a free hypopharynx of its
ancestor. This hypothesis is supported at least by three facts:
(1) the other life-time autogenous genera (Toxorhynchites
and Topomyia) have a free hypopharynx, as well as the par-
tially autogenous species Cx. pipiens molestus and Oc. togoi
showing that even though these mosquitoes do not use their
hypopharynx as blood feeding females did, they still retain
the clue of their common ancestor’s attributes on hypophar-
ynx and other mouthparts such as maxillas and mandibles in
some degrees [1,20], so, the fused hypoharynx of Malaya
should derived from an older attribute of their origin; (2)
geographic distribution of genus Malaya confined only for
Southeast Asia and Papua regions [25,29], showing that this
genus might evolved recently in mosquito evolution and
hence derived their fused hypopharynx from an older mos-
quitoes that had a free hypopharynx as other mosquitoes did;
(3) phylogeny analyses by Harbach and Kitching, using
morphological characters-based cladograms, placed this ge-
nus as the most recent group appeared in mosquito evolution
[32]. The hypothesis suggests that the genus represents the
most advance stage in evolutionary process of the hypophar-
ynx, and that such evolution occurred independently from
other autogeneous genera.
Tribes and genera examined by us are still limited in
view of the recent classification system of mosquitoes by
Harbach and Kitching [32]. Although males of all the species
examined have the hypopharynx fused with the labium, there
are remain possibilities that different states are discovered
for males of the other tribes or genera, especially those put
toward the base of the phylogenic tree of mosquitoes based
on cladistic analyses of Harbach and Kitching [32]. It is also
possible that the hypopharynx fused with the labium is found
in life-time autogenous species yet to be examined. We re-
vealed the diverse states of male maxillae and mandibles
[1,6], and noted that expansion of the scope to all the mos-
quito tribes and genera could contribute to phylogenic study
of mosquitoes. Though the hypopharynx with functions is
much less variable, it could still yield cues to understand the
evolution of feeding habits of mosquitoes.

6 The Open Entomology Journal, 2007, Volume 1 Wahid et al.
ACKNOWLEDGEMENTS
We gratefully thank T. Takahashi, T. Tabata and S. Na-
kahara for their help in the preparation of electron micro-
scope images, I. Miyagi for the suggestions of staining and
dissection methods and providing some samples of Anophe-
les, Toxorhynchites and Tripteroides, H. S. Yong for his help
in mosquito collection in Malaysia, H. Takaoka for his per-
mission to examine some species of black flies (Simulidae)
in his laboratory and Y. Eguchi for providing some of Aedes
and Culex specimens from laboratory colonies. Mosquito
collection in Indonesia was supported by Grant-in-Aid for
Monbusho International Scientific Research Program (Field
Research) (13576009).
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Received: June 16, 2007 Revised: July 11, 2007 Accepted: July 12, 2007