19

Proc. Arthropod. Embryol. Soc. Jpn. 48, 19–32 (2017)Ⓒ 2017 Arthropodan Embryological Society of Japan
ISSN 1341–1527[ORIGINAL PAPER]

Embryonic Development of a Diving Beetle, Hydaticus pacificus Aubé
(Insecta: Coleoptera; Dytiscidae): External Morphology andPhylogenetic Implications*Kazuhiro NIIKURA1, 2), Kei HIRASAWA3, 4), Toshio INODA5, 6)
and Yukimasa KOBAYASHI1, 7)
Department of Biological Sciences, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Minami-ohsawa
1–1, Hachioji-shi, Tokyo 192–0397, Japan
2)
Current address: Forestry Agency, Ministry of Agriculture, Forestry, Fisheries Kasumigaseki 1–2–1, Chiyoda-ku, Tokyo 100–8952,
Japan
3)
Mawatari 81–8, Jyoubanshimofunao-machi, Iwaki-shi, Fukushima 972–8312, Japan
4)
Environmental Aquarium Aquamarine Fukushima, Onahama Pier 2, 50, Tatsumi-cho, Onahama, Iwaki-shi, Fukushima 971–
8101, Japan
5)
Shibamata 5–17–10, Katsushika-ku, Tokyo 125–0052, Japan
6)
Department of Biology, Faculty of Science, Toho University, Miyama 2–2–1, Funabashi, Chiba 274–8510, Japan
7)
Current address: Sayamadai 2–21–18, Sayama-shi, Saitama 350–1304, Japan
E-mail: dineutus@hotmail.co.jp (YK)1)

Abstract
The external features of the egg and developing embryo of the dividing beetle Hydaticus pacificus Aubé are described fromobservations based on light and scanning electron microscopy. The egg period is about 50 h at 28ºC. The newly laid egg is nearlylong ellipsoid, about 2.2 mm long and 0.7 mm wide, and develops to a maximum of 2.7 mm long and 0.9 mm wide. The chorion is thin(about 2 µm thick) and fragile, and has a homogeneous internal structure with a smooth surface. The vitelline membrane beneaththe chorion is tough, and appears to have a potential role of an egg-shell in protecting the egg contents when the chorion is damaged
during the egg period. On the anterior pole, about 35 micropyles have a circular arrangement in the chorion, and the vitelline
membrane just beneath the micropyles forms a large disk-shaped micropylar bulge. The early embryo forms on the ventral side ofthe egg and is wide and long, occupying about three quarters of the egg length. The embryo elongates and widens, retaining thissuperficial position throughout development. Based on our embryological observations of this species, the following features of theegg and embryogenesis are presumed to be unique to the Dytiscidae (diving beetles): 1) The presence of many micropyles with acircular arrangement on the anterior pole of the egg; 2) The temporal appearance of a long intercalary segment in the early embryo;3) The formation of thick longitudinal tracheal trunks accompanied by the closure and disappearance of segmental tracheal
openings, except those of the 8th abdominal segment; 4) Fusion of the 9th and 10th abdominal segments accompanied with extreme
diminution of their size, and their incorporation into the 8th segment during embryogenesis. The phylogeny of Adephaga is
discussed by comparing of the features of eggs and developing embryos among three adephagan families, the Gyrinidae, Carabidae,
and Dytiscidae, and the discussion suggests the non-monophyly of aquatic adephagan families, or Hydradephaga.

Introduction
The Coleoptera, the largest order of Insecta, is composedof four suborders, Archostemata, Myxophaga, Adephaga, andPolyphaga. The second largest suborder, Adepahga, comprises
over 36,000 species in 10 families (Maddison et al., 2009).
Based on their habitats, the families are often categorized into
the terrestrial Geadephaga (Carabidae and Trachypachidae)
and the aquatic Hydradephaga [Amphizoidae, Aspidytidae,

Dytiscidae,
Gyrinidae,
Haliplidae,
Hygrobiidae(=Paelobiidae), Meruidae, and Noteridae] (Crowson, 1960),
but each monophyly of Geadephaga and Hydradephaga ishighly controversial (e.g., Kavanaugh, 1986; Beutel andRoughley, 1988; Maddison et al., 2009).
Despite numerous studies on the embryology of thelargest coleopteran suborder, Polyphaga, that comprises
about 90% species of Coleoptera, embryological information* This article, which was received in 2013 and should have been published in 2014, was printed in 2017 being much delayed due to various circumstances.

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K. NIIKURA, K. HIRASAWA, T. INODA AND Y. KOBAYASHI

is entirely lacking for Archostemata and Myxophaga, and is
scarce for Adepahga, where embryological studies have been
undertaken in only three of the 10 families, Gyrinidae,
Carabidae, and Dytiscidae. In our recent series of comparativeembryological studies of this suborder, the external featuresof the eggs and developing embryos were described in thewhirligig beetle, Dineutus mellyi (Gyrinidae), and the carabid
ground beetle, Carabus insulicola (Carabidae) (Komatsu andKobayashi, 2012; Kobayashi et al., 2013). In the carabidsubfamily Cicindelinae (tiger beetles), the gross embryonicdevelopment of Cicindela togata was reported by Willis (1967).
In the Dytiscidae, however, there are only two old, fragmental
studies on the embryonic development of Dytiscus marginalis(Korschelt, 1912; Blunk, 1914), and thus our embryologicalknowledge of the Dytiscidae is insufficient to understand thefeatures of this family from the standpoint of comparativeembryology.
The present paper is the third report of our comparativeembryological study of Adepahga. It focuses on the external
embryonic features of the diving beetle Hydaticus pacificus
Aubé, a member of the aquatic family Dytiscidae. Based on
the features of eggs and developing embryos of this species,
the phylogenetic relationships of three adephagan families,
the Gyrinidae, Carabidae, and Dytiscidae, are discussed.
Materials and Methods
The eggs of Hydaticus pacificus Aubé, 1838, were
deposited singly on the surface of the floating aquatic fern
Salvinia molesta Mitchell by breeding females in an aquariumfrom July to September of 2007. A total of about 100 eggs wereobtained during this period. Progenitors of the females were
captured at several bogs in Fukushima Prefecture, Japan. The
egg period was about 50 h at a water temperature of 28ºC. The
eggs were removed from S. molesta by fine brushes, and fixedwith alcoholic Bouinʼs fluid every 1 to 3 h after oviposition.
For observations of whole embryos with astereomicroscope, the egg-shells (chorion and vitelline
membrane) were removed with fine forceps and tungsten
needles. For observations of the egg surface by scanningelectron microscopy (SEM), fixed eggs were punctured withtungsten needles, dehydrated by passing through an ethyl
alcohol/isoamyl acetate series and then air dried. For
observations of embryos by SEM, the egg-shells andembryonic membranes (amnion and serosa) were firstremoved from the fixed eggs immersed in 70% ethyl alcoholusing fine forceps, and the fixed embryos were dehydrated
and dried as described for the SEM observations of the egg
surface. Specimens for SEM were coated with gold andobserved using an SEM (JSM-5610, JEOL, Japan).
Results
Egg
The newly laid egg is white and long ellipsoid with aslight convexity at the ventral side on which the prospective
embryo is formed (Fig. 1A). The anterior pole of the egg isslightly more roundish than the posterior pole. The egg isabout 2.2 mm long and 0.7 mm wide, and enlarges to a
maximum of about 2.7 mm long and 0.9 mm wide.
The chorion is thin (about 2 µm thick), soft, andtransparent, and has a homogeneous internal structure with asmooth surface (Fig. 1A, F, ch). The chorion often teared andpeeled away from the egg surface during dehydration for
preparing SEM specimens, exposing the vitelline membranefrom beneath the chorion onto the egg surface (Fig. 1F, vit).
On the anterior pole of the egg, micropyles of about 35 minuteholes are arranged in a circular of about 40 µm in diameter(Fig. 1D, arrows). Just beneath these micropyles, themicropylar area is distinguished as a disk-shaped bulge ofabout 45 µm in diameter and about 10 µm in height (Fig. 1E,mpa). This area is also observed in the eggs with advanced
embryonic development (Fig. 4, mpa).
Embryonic development
Changes in external embryonic features indicate that theegg period (50 h) of H. pacificus is divided into 9 stages, which
can be expressed as a percentage of total developmental time(DT), with 0% at oviposition and 100% at hatching (Bentley etal., 1979). The major developmental events in these stages
are shown in Table 1.
0–8% DT: Maturation of meiotic divisions, fertilization,and cleavage are assumed to occur in the early half of thisstage, but their processes were not observed in the present
study. At about 6% DT, numerous nuclei of blastoderm cells
are observed on the whole egg surface (Fig. 1B, blc).
8–12% DT: A long and wide early embryo, occupying
about three quarters of the egg length, is formed on theventral side of the egg (Fig. 1C). The amnion and serosa areformed by the fusion of amnioserosal folds on the ventral sideof the embryo (Fig. 1C, asf). Towards the end of this stage, theembryo divides into a bi-lobed protocephalon and a longprotocorm (Fig. 1C, pce, pco).
12–24% DT: The posterior end of the embryo elongates
towards the dorsal side of the egg, and thus the embryo isnearly J-shaped in lateral view (Fig. 2A, B). At the posterior
end of the protocephalon, antennary rudiments appear as apair of minute projections (Fig. 2A, at). At the anterior half ofthe protocorm, segmentation of future gnathal and thoracic
regions occurs; i.e., mandibular, maxillary, and labial segments
appear in the former region, and three thoracic segments
appear in the latter (Fig. 2A, mds, mxs, lbs, th1). In the latter
half of this stage, segmentation also begins in the future
abdominal region (Fig. 2C, D). Near the anterior end of the
protocephalon, the stomodaeum appears as a shallow
invagination (Fig. 2C, stom). Although the formation of theprimitive groove is assumed to occur in the previous stage,
the vestige of the groove is observed along the ventral midlineof the protocorm at the beginning of this stage (Fig. 2A, prg),
but it disappears by the end of this stage. Yolk segmentation
occurs in the latter half of this stage.
24–38% DT: The embryo widens, but its length

EMBRYONIC DEVELOPMENT OF HYDATICUS PACIFICUS

somewhat contracts, so that the abdominal end situates at theposterior pole of the egg (Fig. 2E, F). Abdominal segmentation
completes and this region becomes 10-segmented. The
clypeolabral rudiment appears as a large bi-lobed projection
just anterior to the stomodaeum (Fig. 2E, F, cllr). In thegnathal segments, mandibular appendages appear as a pair ofglobose lobes, but maxillary and labial appendages are formed
as a pair of three- and two-segmented projections,
respectively. Late in this stage, the rudiments of galeae, or21

maxillary endites, appear as globose swellings extending
medially in the proximal part of the maxillary appendages(Fig. 2G, ga). In the thoracic segments, the appendages areformed as thick projections developing medio-posteriorly(Fig. 2G, H, th1.l). In the first abdominal segment, pleuropodia
first develop into globose projections (Fig. 2E, F, pp), but later
become flat and cup-shaped (Fig. 2G, H, pp). In themesothoracic and metathoracic segments and the first eight
abdominal segments, tracheal invaginations appear on bothFig. 1 Light micrographs (A–C) and scanning electron micrographs (D–F) of fixed eggs of Hydaticus pacificus. A. Lateral view of a newly
laid egg. B. Lateral view of the egg at 6% DT. C. Ventral view of the egg at 12% DT. D. Anterior pole of a newly laid egg, showing
micropyles (arrows) arranged in a circle. E. Anterior pole of a newly laid egg, showing the micropylar area (mpa) on the vitelline
membrane (vit), and the chorion (ch) peeling away from the micropylar area. F. Egg surface of a newly laid egg, showing the chorionand vitelline membrane; the chorion is partly peeled away from the vitelline membrane. anp: anterior pole, asf: amnioserosal fold,blc: blastoderm cell, pce: protocephalon, pco: protocorm, vnt: ventral side of egg. Scales = A–C: 0.5 mm; D, E: 20 µm; F: 5 µm.
Table 1

Major developmental events according to successive embryonic stages in Hydaticus pacificus. Each stage is expressed as a percentage
of total developmental time (DT), with 0% at oviposition and 100% at hatching, which is taken as 50 h after oviposition.

Stage (DT) (%)

Major developmental event

0–8

Formation of blastoderm

8–12

Formation of embryo with protocephalon and protocorm. Formation of amnioserosal fold and completion of embryonicmembranes

12–24

Elongation of embryo accompanied by commencement of segmentation. Formation of stomodaeum

24–38

Completion of segmentation. Differentiation of labrum. Appearance of gnathal and thoracic appendages. Formation of proctodaeal
invagination. Formation of tracheal invaginations

38–52

Elongation of gnathal and thoracic appendages. Closure of openings of tracheal invaginations except those of the 8th abdominal
segment. Fusion of the 9th and 10th abdominal segments

52–66

Rupture of embryonic membranes and progress of provisional dorsal closure. Formation of secondary dorsal organ. Katatrepsis
or postural change with ventrally flexed abdominal end. Appearance of urogomphi

66–80

Completion of definitive dorsal closure. Establishment of basic form of the first instar larva. Embryonic molt

80–92

Completion of thick longitudinal tracheal trunks

92–100Establishment of definitive first instar larval form with egg teeth in head capsule

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K. NIIKURA, K. HIRASAWA, T. INODA AND Y. KOBAYASHI

Fig. 2 Successive stages of embryonic development of Hydaticus pacificus. The chorion is omitted in all figures. A. About 14% DT (ventralview). B. About 14% DT (lateral view). C. About 20% DT (ventral view). D. About 20% DT (lateral view). E. 30% DT (ventral view).
F. 30% DT (lateral view). G. 38% DT (ventral view). H. 38% DT (lateral view). ab1,8,9,10: 1st, 8th, 9th, and 10th abdominal segment.
am: amnion, at: antenna, cllr: clypeolabrum, ga: galea, int: intercalary segment, lb: labium, lbs: labial segment, md: mandible, mds:
mandibular segment, mx: maxilla, mxs: maxillary segment, ngr: neural groove, pce: protocephalon, pp: pleuropodium, prg:
primitive groove, proct: proctodaeum, se: serosa, stom: stomodaeum, th1: prothoracic segment, th1.l: prothoracic leg, th3.l:
metathoracic leg, tri: tracheal invagination. y: yolk. Scales = 1 mm.

sides of each segment (Fig. 2E, F; 4, tri). The neural groove
appears along the ventral midline of the germ band (Fig. 2E–
H, ngr). The small proctodaeal invagination is observed at theabdominal end (Fig. 2G, proct).
In the SEM micrograph showing the embryo at about

30% DT, the intercalary segment is clearly observed as a long(about 110 µm) segment without appendages, just anterior tothe mandibular segment (Fig. 4, mds, int). In the anterior halfof the intercalary segment, a pair of low swellings is observed(Fig. 4, arrow). These swellings are assumed to represent toEMBRYONIC DEVELOPMENT OF HYDATICUS PACIFICUS

23

Fig. 3 Successive stages of embryonic development of Hydaticus pacificus. The chorion is omitted in all figures. A. 48% DT (ventral view).
B. 60% DT (ventral view). C. 60% DT (lateral view). D. 72% DT (ventral view). E. 96% DT (ventral view). F. 96% DT (lateral view).
ab1,8,9,10: 1st, 8th, 9th, and 10th abdominal segment, at: antenna, cllr: clypeolabrum, et: egg tooth, gng: ganglion, hyp: hypopharynx,
lb: labium, lbp: labial palp, md: mandible, mx: maxilla, mxp: maxillary palp, pp: pleuropodium, sdo: secondary dorsal organ, spr:
spiracle, stom: stomodaeum, suc: sucking channel, th1.l: prothoracic leg, trt: tracheal trunk, ur: urogomphus. Scales = 1 mm.

the ganglion mother cells. In the abdominal segments, theopenings of the tracheal invaginations of the 7th and 8th
segments assume an L-shape and are larger than those ofother segments (Fig. 4, tri).
38–52% DT: Early in this stage, the integration of the

cephalognathal region advances (Fig. 3A), i.e., the clypeolabralrudiment becomes flat and extends posteriorly, and the labial
appendages on both sides shift medially and fuse to form alabium. The mandibular appendages slightly extend medioposteriorly, and the maxillary and labial appendages also

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K. NIIKURA, K. HIRASAWA, T. INODA AND Y. KOBAYASHI

extend medio-posteriorly and differentiate their palpi. The
thoracic appendages further elongate and their tips attain to
the ventral midline. By the end of this stage, the openings oftracheal invaginations close and disappear in the mesothorax,
metathorax and the first seven abdominal segments, but the
openings in the 8th abdominal segment remain open as
spiracles (Fig. 3A, spr). The neural groove is obliterated, andthe newly formed ganglia of the ventral nerve cord areobserved through a thin ectodermal layer (Fig. 3A, gng). At
the abdominal end, the 9th and 10th abdominal segments are
fused into one segment accompanied with diminution of size
(Fig. 3A, ab9, ab10), and thus the abdomen seemingly consistsof 9 segments.
52–66% DT: Katatrepsis or the postural changes of theembryo proceeds (Fig. 3B, C). In this process, the posterior
abdominal segments turn ventrally, directing the abdominal
end anteriorly. At the abdominal end, the fused 9th and 10th
segments are further reduced and integrated into the ventroposterior side of the large 8th segment so that the abdomen
seemingly consists of 8 segments, and urogomphi appear as apair of stout projections on the ventral side of the abdominalend (Fig. 3B, C, ur). The integration of the cephalognathal
region further proceeds and the basic form of the larval headis completed; the antennae, maxillary and labial palpi elongate
posteriorly, and the mandibles elongate and become sickleshaped. The basal region of the fused labium forms the
hypopharynx (Fig. 3B, hyp), shifts anteriorly between the
maxillae and almost touches the clypeolabrum. The thoracic
appendages extremely elongate posteriorly and the tips of themeso- and metathoracic appendages almost reach theposterior pole of the egg.
Accompanying katatrepsis, the embryonic membranes(amnion and serosa) fuse at the ventral side of the embryo,and rupture at this point to form an amnioserosal fold. The
embryonic membranes then regress on the dorsal surface ofthe yolk mass where the concentrated serosa forms a large
secondary dorsal organ (Figs. 3B, C; 5A, sdo, asf), and theinverted amnion provisionally covers the dorsal surface of theyolk mass (provisional dorsal closure) (Fig. 5A, am). The
anterior region of the secondary dorsal organ temporarily
assumes a cap-like structure on the dorsal side of theembryonic head (Fig. 3B, C, sdo).
66–80% DT: Early in this stage, the lateral walls of theembryo extend dorsally, causing the yolk mass to becompletely enclosed by the dorsolateral walls so that thedefinitive dorsal closure is completed. After dorsal closure,the basic form of the first instar larva is established (Fig. 3D);
the antennae, maxillary and labial palpi are three-segmented,
and the thoracic appendages become five-segmented. The
clypeolabrum becomes flat and wide. Urogomphi elongate andbecome slender (Figs. 3D; 5B, C, ur). At the ventral side of theabdominal end, the fused 9th and 10th abdominal segments
assume as a minute swelling between the basal parts ofurogomphi (Fig. 5C, ab9+10).
At about 70% DT, a delicate membrane, or embryonic

Fig. 4 Scanning electron micrograph of the Hydaticus
pacificus embryo (ventral view) at 30% DT. The
amnion and serosa are removed, and the rightprothoracic leg accidentally destroyed during
preparation of the specimen for SEM. ab1,9: 1st and9th abdominal segment, at: antenna, cllr:
clypeolabrum, int: intercalary segment, lb: labium,
lbs: labial segment, md: mandible, mds: mandibular
segment, mx: maxilla, mxs: maxillary segment, mpa:
micropylar area on vitelline membrane, ngr: neuralgroove, pp: pleuropodium, stom: stomodaeum, th1:
prothoracic segment, th1.l: prothoracic leg, tri:
tracheal invagination, y: yolk, arrow: ganglion
mother cells. Scale = 200 µm.

EMBRYONIC DEVELOPMENT OF HYDATICUS PACIFICUS

25

Fig. 5 A. Light micrograph of the Hydaticus pacificus egg (dorsal view) at 60% DT, showing the secondary dorsal organ (sdo) andprovisional dorsal closure by the inverted amnion (am). The chorion is removed. B. Light micrograph of the Hydaticus pacificus egg(ventral view) at 72% DT, showing the embryonic cuticle (arrows) which detaches from the whole body wall. C. Scanning electron
micrograph of the abdominal end (ventral view) of the embryo at 72% DT, showing the developing urogomphi (ur) and the embryoniccuticle (arrows) which peels away from the body wall. The micrograph shows the ventral view of the abdominal ends whichcorresponds to the area of rectangle in B. D. Light micrograph of the newly hatched larva (lateral view) of Hydaticus pacificus.
ab1,7,8,10: 1st, 7th, 8th, 9th, and 10th abdominal segment, asf: amnioserosal fold, et: egg tooth, hc: head capsule, lw: lateral wall, th1:
prothoracic segment, trt: tracheal trunk. Scales = A, B: 0.5 mm; C: 100 µm; D: 1 mm.

cuticle, is often observed to slightly detach from the wholebody wall (Fig. 5B, arrows). The SEM micrograph of this stage
also shows fragments of the embryonic cuticle peeling awayfrom the larval cuticle (Fig. 5C, arrows). These observations
suggest that the embryonic molt occurs around this stage.
80–92% DT: The egg rapidly enlarges to about 2.7 mm
long and 0.9 mm wide. A pair of thick longitudinal tracheal

trunks appears as brown tubes through the semi-transparent
integument on both sides of the embryo (cf. Figs. 3E, F; 5D,
trt). The two tracheal trunks run from the prothorax to theposterior end of the 8th abdominal segment, where thetracheal trunks open as a pair of large spiracles.
92–100% DT: The definitive form of the first instar larva
completes and hatching occurs at about 50 h after oviposition

26

K. NIIKURA, K. HIRASAWA, T. INODA AND Y. KOBAYASHI(Fig. 3E, F). In the medial part of the mandibular distal half,
the sucking channel is observed as a thin dark-brown tube
(Fig. 3E, suc). On the frons, a pair of brown sclerotized spines,
or egg teeth, appears at about 95% DT (Fig. 3F, et). The egg
teeth are retained on the head of the hatched larva (Fig. 5D,et). The pleuropodia gradually degenerate and disappear by
the time of hatching. At hatching, the larval head first ruptures
the anterodorsal part of the egg-shells (chorion and vitelline
membrane), and the larval thorax and abdomen throw off theegg envelopes. The embryonic cuticle, which has alreadydetached from the embryonic body wall at the time of theembryonic molt (about 70% DT), remains on the inner surfaceof the vitelline membrane.
Discussion
Egg
The chorion of Hydaticus pacificus is thin and soft (about
2 µm thick), and has a homogeneous internal structure with asmooth surface (Fig. 1A, F, ch). Although the chorion does nottear under natural conditions, it often peels away from the egg
surface during dehydration for preparing SEM specimens. In
such a case, the vitelline membrane beneath the chorion is
exposed on the egg surface, even in newly laid eggs (Fig. 1E,F, vit). Thus, the vitelline membrane appears to have apotential role in protecting the egg throughout the egg period.
In the newly laid eggs of another dytiscid species, Dytiscus
marginalis, Blunck (1914) observed two discrete membranes,
the chorion and vitelline membrane, with almost the same
thickness, although the author did not specify the thickness.
As the egg enlarges long before hatching, the chorion on both
the dorsal and ventral sides of the egg splits longitudinally(Blunck, 1914; Jackson, 1959) in contrast to H. pacificus.
Moreover, the thick serosal cuticle is formed beneath thevitelline membrane by the secretion of the serosal cells, andthus both the vitelline membrane and serosal cuticle
practically protect the egg contents (Blunck, 1914; Jackson,1959). The presence of a thin and fragile chorion and a toughvitelline membrane is also reported in the eggs of anotherdytiscid species, Agabus bipustulatus, although the split of thechorion does not occur in this species in natural conditions(Jackson, 1958).
In the newly laid egg of the carabid ground beetle
Carabus insulicola, the chorion is also thin and soft, but has amesh-like chorionic structure on its surface (Kobayashi et al.,2013). This is also the case for other carabid beetles (Luff,
1981; Kaupp et al., 2000). Conversely, in the aquatic family
Gyrinidae the eggs have a relatively thick chorion (e.g., about
20 µm thick in Dineutus mellyi, Komatsu and Kobayashi,2012), and their surface is either covered in blunt conicalprojections (Dineutus horni, Baker and Ma, 1987; Di. mellyi,
Komatsu and Kobayashi, 2012) or has a sculptured, ribbed, orhoneycombed structure (Gyrinus mirinus and Gyrinus. sp.,
Hinton, 1981). Moreover, in both C. insulicola and Di. mellyi,
the hard serosal cuticle (e.g., 4 to 5 µm thick in C. insulicola)
completes beneath the chorion at about 20% DT. In C.

insulicola, enlargement of the egg during development causes
the soft chorion to stretch and, occasionally, to peel away fromthe serosal cuticle (Kobayashi et al., 2013). In Di. mellyi, as thesize of the egg increases, the tough thick chorion splits
longitudinally along the ventral midline well before hatching(Komatsu and Kobayashi, 2012). Therefore, as in other gyrinid
species, the newly formed serosal cuticle beneath the chorion
actually plays the role of an egg-shell protecting the egg
contents after the splitting of the chorion.
In summary, the fragile character of the chorion and the
major role played by the vitelline membrane in protecting theegg contents must be features that are phylogeneticallyconstrained for all eggs of the Dytiscidae. In H. pacificus, ahard and tough serosal cuticle like that of C. insulicola, Di.
mellyi, and Dy. marginalis is not detected by external
observations throughout the egg period, although thepossibility of the formation of a very thin serosal cuticle
undetectable by external observations is not denied. Thus,
the degree of development of the serosal cuticle may vary
according to the taxa within the Dytiscidae.
In H. pacificus, about 35 micropyles (Fig. 1D, arrows) arearranged in a circle of about 40 µm in diameter around theanterior pole of the egg. In the eggs of other dytiscid species,
many micropyles are also arranged in a circle, such as about 60
in Dy. marginalis (Blunck, 1914) and about 17 in A. bipustulatus(Jackson, 1958). On the other hand, in the eggs of all gyrinid
species examined to date, the micropyles are present at the
top of the stalk-like projection situated at the anterior pole ofthe egg (Saxod, 1964; Hinton, 1981; Baker and Ma, 1987;
Komatsu and Kobayashi, 2012). Thus, the Dytiscidae andGyrinidae are strikingly different, not only in the thickness ofthe chorion but also in the structures of the micropylar region.
In the eggs of the suborder Polyphaga, information about
the detailed structures of micropyles is scarce (Hinton, 1981).
However, in the several species whose micropyles have been
examined, many micropyles are arranged almost in a circle;
e.g., the numbers of micropyles are 16 to 24 in Lytta viridana(Meloidae) (Sweeny et al., 1968), about 10 in Rhagophthalmus
ohbai (Rhagophthalmidae) (Kobayashi et al., 2002), about 15 inPrionotolytta binotata (Meloidae) (Bologna and Di Giulio,
2003), and about 20 in Hermonia axyridis (Osawa andYoshinaga, 2009). While there are considerable variations in
the number of micropyles, their circular arrangement near
the anterior pole seems to be a character common to
Polyphaga and Adephaga excluding the Gyrinidae.
In H. pacificus, just beneath the circularly arranged
micropyles in the chorion, the micropylar area on the vitelline
membrane is present as a disk-shaped bulge (Fig. 1E, mpa). A
similar disk-shaped area is also detected on the vitelline
membrane in the anterior pole of the egg of Dy. marginalis(Jackson, 1959). It is presumed that this area functions as thepassages through which spermatozoa enter the egg, but thedetailed internal structure of this area remains unknown.

EMBRYONIC DEVELOPMENT OF HYDATICUS PACIFICUS

27

Growth pattern of embryo
The early embryo of H. pacificus, like that of Dy.
marginalis (Korschelt, 1912; Blunck, 1914), is formed on theventral side of the egg, and it subsequently develops on theegg surface throughout the egg period. Thus, the embryo
belongs to the superficial type found in many holometabolous
insects. The embryos of the gyrinid Di. mellyi, and thecarabids Carabus insulicola and Cicindela togata also belong to
the superficial type (Komatsu and Kobayashi, 2012; Kobayashi
et al., 2013; Willis, 1967). Therefore, it is presumed that theembryos of adephagan families probably belong to thesuperficial type. On the other hand, although most embryos of
the polyphagan Coleoptera belong to the superficial type (e.g.,
Lytta viridana, Rempel and Church, 1969), in some families
such as the Lampyridae (fireflies), Rhagophthalmidae(glowworms), and some Chrysomelidae (leaf beetles), theearly embryos are formed by deep invagination of the germdisks into the yolk (Williams, 1916; Miya, 1965; Zakhvatkin,1967, 1968; Ando and Kobayashi, 1975; Krysan, 1976;
Kobayashi and Ando, 1985; Kobayashi et al., 2002, 2006). In
particular, embryos of the Lampyridae and Rhagophthalmidae
are differentiated from the spherical germ rudiments, which
are formed by deep invagination of the small circular germdisks into the yolk, and the embryos then develop within theyolk in the submerged condition until close to katatrepsis(Ando and Kobayashi, 1975; Kobayashi and Ando, 1985;
Kobayashi et al., 2002, 2006). Thus, the growth pattern of thepolyphagan embryos is specialized or diversified in somegroups, whereas that of the adephagan embryo is probably
uniform throughout the suborder.

thereafter. In H. pacificus, however, the intercalary segment
remains clearly observable externally, even at around 30% DT
when the pleuropodia appear (Fig. 4). Thus, the period of
appearance of this segment is exceptionally long in the H.
pacificus embryo, and this character is unique to this species.
In the H. pacificus embryo, the galea is formed as a small,medially extended endite lobe at the proximal part of themaxillary appendage, and another endite corresponding to thelacinia is not formed (Fig. 2G, H, ga). In the embryos of Di.
mellyi and C. insulicola, both the lacinia and galea arise as
separate endite lobes at the proximal part of the maxillary
appendages, and are retained in the larval stages, although thelacinia in the C. insulicola larva is highly vestigial (Komatsu
and Kobayashi, 2012; Kobayashi et al., 2013). Beutel (1993)
postulated that the absence of the lacinia from larvae of theTrachypachidae and the superfamily Dytiscoidea (Noteridae,
Amphizoidae, Hygrobiidae, and Dytiscidae) is a synapomorphyof these groups, suggesting the sister group relationship ofthem. Alarie and Bilton (2005) also confirmed the absence ofthe lacinia in the larvae of another dityscoid family
Aspidytidae. In most non-holometabolous insects, both thelacinia and galea arise as endites of the maxillary appendage inthe embryonic stage, but in some groups of Holometabola(e.g., Trichoptera, Kobayashi and Ando, 1990) the lacinia is notformed in the embryos. The appearance of both the lacinia andgalea in Di. mellyi and C. insulicola in their embryonic stages
is thus regarded as the primitive state, and the absence of
lacinia in H. pacificus as the derived one. These embryologicalresults support Beutelʼs postulation that the loss of lacinia
should be an apomorphic feature.

Cephalognathal region
In the H. pacificus embryo at about 30% DT, theintercalary segment is clearly observed as a long segment justanterior to the mandibular segment (Figs. 2E; 4, int). Theintercalary segment is somewhat longer than the mandibular
segment in this stage, although the former becomesundetectable in the next stage (Fig. 2G, H). In insect
embryogenesis generally, the intercalary segment is shortand appears temporarily in the early stage when thesegmentation of the cephalognathal region commences, andthe segment soon disappears because it merges into the
cephalic lobe. Thus, the presence of the intercalary segment
is often overlooked by external observations, particularly inholometabolous insect embryos, and is detected only byhistological observations (e.g., Lytta viridana in Coleoptera,
Rempel and Church, 1969). However, in a non-holometabolous
insect, such as Galloisiana yuasai (Grylloblattodea), the large
intercalary segment is formed in the young embryo (Uchifune
and Machida, 2005). In Adephaga, the intercalary segment isalso temporarily observed in the young embryos of Di. mellyi(Gyrinidae) and C. insulicola (Carabidae) (Komatsu andKobayashi, 2012; Kobayashi et al., 2013). In both species, theintercalary segment appears at 10 to 20% DT, or before theappearance of pleuropodia, and becomes undetectable

Abdominal segmentation
Among adephagan families, the larvae of the Gyrinidae,
Carabidae, and Trachypachidae have a 10-segmented
abdomen (Arndt et al., 2005; Beutel, 2005; Beutel and Arndt,
2005; Beutel and Roughley, 2005). Embryological observations
revealed that the larval 10th abdominal segment in the
Gyrinidae and Carabidae is formed by the fusion of theembryonic 10th and 11th segments and non-segmental telson
in which the proctodaeal invagination is formed (Komatsu andKobayashi, 2012; Kobayashi et al., 2013).
In other adephagan families, excluding the Haliplidae,
and in the superfamily Dytiscoidea, the larval 9th and 10th
abdominal segments are strongly reduced or entirely lacking(Vondel, 2005; Dettner, 2005a, b, c; Balke, 2005). Extreme
reduction of abdominal segments is found in dytiscid larvae,
where the abdomen of the fully grown larva consists of 8
segments with paired urogomphi on the abdominal end,
although urogomphi are absent in Cybister (Kamite, 2008). In
Dy. marginalis, however, both Korschelt (1912) and Blunck(1914) showed that the number of abdominal segments in theold embryo is 9, instead of 11 (excluding the telson), in theyoung embryo. Korschelt (1912) attributed the fewer
abdominal segments in the old embryo to reduction (or loss)of the posterior segments. The present study revealed that

28

K. NIIKURA, K. HIRASAWA, T. INODA AND Y. KOBAYASHI

the 9th and 10th abdominal segments form in the young
embryo of H. pacificus, but did not detect the 11th segment byexternal observations (Fig. 2E–H, ab9, ab10). In this species,
as katatrepsis comes close, the 9th and 10th segments are
fused into one segment with the latter being diminutive insize (Fig. 3A, ab9, ab10). After katatrepsis, the fused 9th and10th segments are further reduced and incorporated into theventro-posterior part of the 8th segment, so that the abdomen
becomes seemingly composed of 8 segments with pairedurogomphi (Fig. 3B–F, ab8, ur). The fused 9th and 10th
segments are observed at the ventro-posterior end of the 8thsegment, or between the bases of urogomphi (Fig. 5C, ab9+10). Thus, the larval 8th abdominal segment is formed byincorporation of the embryonic 9th and 10th segments into
the 8th segment, not by the complete loss of the 9th and 10th
segments.
The present study could not trace the detailed process ofthe formation of urogomphi of H. pacificus. Snodgrass (1931)
thought that the urogomphi of coleopteran larvae are merely
an integumentary outgrowth of the 9th tergum. Conversely,
Matsuda (1976) assumed that coleopteran larval urogomphi
are homologous with cerci often arising at the 11th abdominal
segment in primitive insects such as Archaeognatha. He
further considered that the urogomphi of dytiscid larvae arise
from the posterolateral angles of the 8th segment where theembryonic 11th segment has been incorporated. In the
carabid ground beetle C. insulicola, however, detailed
embryological observations revealed that the urogomphi
originate in the 9th tergum (Kobayashi et al., 2013), thus
supporting Snodgrassʼ view. As mentioned before, the present
study on H. pacificus, like the studies on Dy. marginalis by
Korschelt (1912) and Blunck (1914), could not clarify the exact
positions from which the urogomphi arise. However, it isunlikely that the urogomphi of H. pacificus originate in the
11th segment as presumed by Matsuda (1976), because thesegment cannot be found during embryogenesis, or if thesegment is temporarily present, it must be extremely
vestigial and undetectable from external observations.
Instead, in the light of the 9th tergal origin of the urogomphi in
C. insulicola, it may be possible to consider the origin ofurogomphi of H. pacificus being also in the 9th segment, whichis incorporated into the 8th segment during embryogenesis.
Tracheal invaginations and longitudinal tracheal trunks
The first instar larva of H. pacificus, like that of otherdytiscid species, has a pair of spiracles near the posterior endof the 8th abdominal segment; thus, despite its aquatic habitat,
the larva breathes air through the spiracles by raising theabdominal end to the water surface periodically. In theembryonic period, however, tracheal invaginations are formedin the meso- and metathoraxes and the first eight abdominal
segments at about 30% DT (Figs. 2E, F; 4, tri). In insect
embryogenesis in general, the bottom of each trachealinvagination forks, and the posterior branch of one fork fuseswith the anterior branch of the next one, thereby forming two

major longitudinal tracheal trunks. It is thus presumed thatthe thick longitudinal tracheal trunks in the fully grownembryo or the first instar larva of H. pacificus (Figs. 3E, F; 5D,
trt) likewise complete by fusion of the anterior and posterior
branches of tracheal invaginations. In this species, however,
the tracheal openings, excluding those of the 8th abdominal
segment, close and disappear at about 50% DT, or before
katatrepsis (Fig. 3A). The completed tracheal trunks of thisspecies, probably as well as those of other dytiscid larvae, aremuch thicker than those of terrestrial insect larvae. This may
be relevant to that dytiscid larvae, which submerge in thewater for a long time, need to keep a large amount of air in the
tracheal trunks. The closure of tracheal openings, excluding
those of the 8th segment, is also regarded as an adaptation to
the aquatic life style of the Dytiscidae, and probably occurs
during embryogenesis of other Dytiscoidea (Hygrobiidae,
Noteridae, Aspidytidae, and Amphizoidae) whose first instar
larvae have functional spiracles only in the 8th abdominal
segment. In contrast to these Dytiscoidea, the first andsecond instar larvae of the Gyrinidae have no spiracle;
instead, they have 10 pairs of long tracheal gills in theabdomen (Beutel and Roughley, 2005). In the embryonicperiod of the gyrinid Di. mellyi, however, 10 pairs of trachealinvaginations (two thoracic and eight abdominal) are formed,
but these openings close as the tracheal gills grow before
katatrepsis, and so the newly hatched larva has no spiracle
and breathes through the gills (Komatsu and Kobayashi,2012). It has been known that the closure and disappearance of
all tracheal openings in the embryonic period occurs in thefollowing orders, all of which have larvae that are completely
aquatic: Odonata (Ando, 1962); Megaloptera (Miyakawa, 1979;
Ando et al., 1985); and Trichoptera (Kobayashi and Ando,
1990). In this aspect, the first and second instar larvae of Di.
mellyi, whose all embryonic tracheal openings close before
katatrepsis, are completely aquatic as those of the aquaticorders mentioned above. Therefore, from an embryologicalstandpoint it can be interpreted that the degree of adaptation
to the aquatic life style in the Gyrinidae is far stronger than in
the Dytiscidae (or the Dytiscoidea).
Embryonic molt and egg teeth
In the H. pacificus embryo, embryonic molt occurs ataround 70% DT, or several hours after the definitive dorsal
closure completes. The embryonic cuticle detaches from thewhole body surface (Fig. 5B, arrows), and at hatching the shedembryonic cuticle remains on the inner surface of the vitelline
membrane. In the eggs of other dytiscid species, Dy.
marginalis and A. bipustulatus, Jackson (1957) observed theshed embryonic cuticle on the surface of fully grown embryos
near hatching, but the exact timing of the embryonic molt was
not specified. An embryonic molt was also suggested in twoother adephagan species, Di. mellyi and C. insulicola, but theexact timing and fate of the shed embryonic cuticle were
obscure (Komatsu and Kobayashi, 2012; Kobayashi et al.,2013). Thus, the present study provides the first examples in

EMBRYONIC DEVELOPMENT OF HYDATICUS PACIFICUS

which both the exact time of embryonic molt and the fate of
the shed embryonic cuticle have been shown in Adepahga.
In H. pacificus, egg teeth appear as a pair of minutesclerotized projections on the frons of the head of the fullygrown embryo at 95% DT (Fig. 3F, et), or at the stage wellafter embryonic molt. The egg teeth are retained during thefirst instar larval stage (Fig. 5D, et). The presence of paired
egg teeth (egg-bursters) in newly hatched larvae has beenreported in many other dytiscid species (e.g., as in Emden,
1925, 1946; Jackson, 1957, 1958) and other dytiscoid families
Amphizoidae (Xie, 2000), Hygrobiidae (Alarie et al., 2004), andAspidytidae (Alarie and Bilton, 2005). In the carabid groundbeetle C. insulicola, a pair of egg teeth also arises on the headof the fully grown embryo, and they are retained in the newly
hatched larva (Kobayashi et al., 2013). In the gyrinid Di. mellyi,
however, neither the embryo nor larva has egg teeth(Komatsu and Kobayashi, 2012).
In insects, the presence of teeth has been reported in 22
of 29 dicondylian orders including the apterygote Zygentoma,and egg teeth are classified into several types according to thelocations where they are formed and the period when they
exist (Kobayashi, 2016). In most orders, other thanColeoptera, a single egg tooth is formed as a minutesclerotized projection in the serosal cuticle covering thefrontal region of the head at the last embryonic stage. At
hatching, when the serosal cuticle is shed off from the larval
cuticle, the egg tooth also is peeled from the larval cuticle ofthe head, and consequently the hatched larva has no egg
tooth. In some of the adephagan Coleoptera, however, as
mentioned before, egg teeth appear as a pair of projections on
both sides of the frons near the frontal sutures. Furthermore,
the adepahgan egg teeth exist in the first instar larval stage,or persist even in the second instar stage in the Aspidytidae(Alarie and Bilton, 2005). Thus the paired egg teeth of
Adephaga are probably not homologous with a single egg
tooth in other orders, which suggests the paired egg teeth
being autapomorphic structures acquired within a lineage of
Adephaga. Beutel (1993) assumed that the presence of egg
teeth in first instar larvae is an autapomorphy of Adephaga,excluding the Gyrinidae and Haliplidae. Thus, theembryological data obtained to date, though scarce, are notcontradictory to Beutelʼs assumption.
Phylogenetic considerations
Crowson (1960) kept the traditional classification system
in which Adephaga are divided into two main sections,Geadephaga and Hydradephaga. However, Crowsonʼsapproach was a pre-cladistic method, and most of thesubsequent cladistics approaches by many authors questioned
whether Geadephaga and Hydradephaga are eachmonophyletic. For example, based on various larval and adultcharacters, Beutel and Roughley (1988) first proposed that theaquatic family Gyrinidae is the sister group of the remaining
adephagan families, in which the Rhysodidae (the carabidsubfamily Rhysodinae) is the sister group of the remaining

29

families; the result suggested that Geadephaga andHydradephaga are each non-monophyletic. In anotheranalysis by Beutel (1993) based on larval head characters, theGyrinidae also occupied the basal position of Adephaga, but of
the remaining families the aquatic family Haliplidae occupied
the basal position. In both studies, the aquatic families
Dytiscidae, Hygrobiidae, Amphizoidae, and Noteridae form amonophyletic unit, or the Dytiscoidea, forming the sistergroup of the terrestrial family Trachypachidae. On the other
hand, in recent years, several molecular studies designed toinfer the phylogenetic relationships among the adephaganfamilies recovered a monophyletic Geadephaga and amonophyletic Hydradephaga (e.g., Ribera et al., 2002; Hunt etal., 2007). However, the most recent molecular phylogenetic
analysis by Maddison et al. (2009) supported only themonophyletic Geadephaga, but not the monophyly of
Hydradephaga, because the analysis suggested that only the
Gyrinidae is the sister group of Geadephaga. As a result, thevalidity of Geadephaga and Hydradephaga in the adephagan
phylogenetic system remains debatable.
As mentioned in the ʻIntroductionʼ, embryologicalinformation has been accumulated in only three of the 10
adephagan families (Gyrinidae, Carabidae, and Dytiscidae),and thus is not yet sufficient for discussing the phylogeny of
the whole Adephaga. However, through comparisons of theexternal features of the eggs and developing embryos among
these three families (as partly discussed above), the following
phylogenetic considerations can be applied to part of
Adephaga. Although the superficial-typed embryos arecommon in these three families, the aquatic families Gyrinidae
and Dytiscidae are entirely different in the following
characters. Character 1: The presence of a stalk-likemicropylar region in the eggs of the Gyrinidae(autapomorphic), whereas the presence of many micropyles
in a circular arrangement in the eggs of the Dytiscidae(probably synapomorphic, shared with Polyphaga). Character
2: A thick chorion with a complicated internal structure and asculptured surface in the Gyrinidae (auapomorphic), whereasa thin and fragile chorion with a smooth surface in the
Dytiscidae (probably synapomoprhic, shared with the
Carabidae). Character 3: The temporary appearance of a shortintercalary segment in the Gyrinidae (plesiomorphic,
observed in many other insects including the Carabidae),whereas the appearance of a long intercalary segment whose
period of existence also is long in the Dytiscidae(autapomorphic). Character 4: The fusion of the embryonic
10th and 11th abdominal segments to form the larval terminal
abdominal segment in the Gyrinidae (symplesiomorphic,
shared with the Carabidae), whereas the fusion of the 8th to10th segments to form the terminal segment in the Dytiscidae(probably autapomorphic). Character 5: The formation of boththe lacinia and galea in the maxillary appendage in the
Gyrinidae (plesiomorphic, also observed in the Carabidae andmany non-holometabolous insects), whereas the absence of
lacinia in the Dytiscidae (apomorphic, often observed in many

30

K. NIIKURA, K. HIRASAWA, T. INODA AND Y. KOBAYASHI

other holometabolous insects). Character 6: The absence ofurogomphi on the 9th abdominal segment in the Gyrinidae(plesiomorphic), whereas the formation of urogomphi
probably originating in the 9th abdominal tergum in the
Dytiscidae (synapomorphic, shared with the Carabidae andprobably other adephagan families excluding the Gyrinidae).
Character 7: The formation of 10 pairs of abdominal tracheal
gills accompanied with the closure of all tracheal openings in
the Gyrinidae (autapomorphic), whereas the formation of
thick longitudinal tracheal trunks accompanied with theclosure of tracheal openings except those of the 8th abdominal
segment on which large spiracles open in the Dytiscidae(autapomorphic). Character 8: The absence of the egg tooth in
the Gyrinidae (plesiomorphic), whereas the formation ofpaired egg teeth on the frons in the Dytiscidae (synapomorphic,
shared with the Carabidae and probably all other adephaganfamilies excluding the Gyrinidae).
In summary, there is no embryonic synapomorphy
between the Gyrinidae and Dytiscidae, which means that
there is no positive embryological data supporting themonophyly of Hydradephaga to date. Conversely, threeapomorphic characters (Characters 2, 6, and 8) are shared
between the Carabidae and Dytiscidae, suggesting an affinityof these families, which means the non-monophyly of
Hydradephaga, although the Carabidae retains someplesiomorphic characters (Characters 3, 4, and 5) shared with
the Gyrinidae. To discuss the phylogeny of the whole
Adepahga from embryological standpoints, further
embryological studies on other adephagan families areneeded, particularly on the aquatic family Haliplidae and theterrestrial family Trachypachidae.

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