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Proc. Arthropod. Embryol. Soc. Jpn. 54, 1–9 (2022)Ⓒ 2022 Arthropodan Embryological Society of Japan
ISSN 1341–1527

Paranotal Lobes Are Appendicular in Origin:
Elucidation by Micro-CT Analysis of the Thoracic Muscular Systemin the Larvae of Carabus insulicola (Insecta, Coleoptera)
Yukimasa KOBAYASHI1), Heather S. BRUCE2), Karen TERADA3),
Akihiro HIRAYAMA4), Yuuki OOSAWA5), Kazuhiro NIIKURA6)
and Yasuoki TAKAMI3)
Sayamadai 2–21–18, Sayamashi, Saitama 350–1304, Japan
Patel Laboratory, Marine Biological Laboratory, 7MBL Street, Woods Hole, MA 02543, USA
3)
Graduate School of Human Development and Environment, Kobe University, Nada, Kobe 657–8501, Japan
4)
Hyogo Prefectural Institute of Technology, Suma, Kobe 654–0037, Japan5)
Toyogaoka 2–1–10–302, Tama-shi, Tokyo 206–00341, Japan
6)
Forestry Agency, Ministry of Agriculture, Forestry and Fisheries, Kasumigaseki 1–2–1, Chiyoda-ku, Tokyo 100–8952,
Japan
E-mail: dineutus@hotmail.co.jp (YK)1)
2)

Abstract

In the embryo of Carabus insulicola, Kobayashi et al. (2013: J. Morphol., 274, 1323–1352) revealed that thesubcoxa can be divided into subcoxae-1 and 2 by the paracoxal suture (PCXS), and these subcoxae form the larval
pleuron. Mashimo and Machida (2017: Sci. Rep., 7, 12597), however, rejected the appendicular nature of subcoxa-1 andinterpreted the PCXS as the boundary between the tergum and appendage (BTA), and thus they regarded subcoxa-1
as a tergal structure. To resolve the disagreement over the PCXS, we examined the larval thoracic muscular system
of C. insulicola using an X-ray micro-CT. We conclude that subcoxae-1 and 2 are independent podomeres having
intrinsic muscles in each region and that the paranotal lobe of the C. insulicola larvae is derived from the dorsal halfof subcoxa-1. Thus, the paranotal lobe is appendicular in origin, contradictory to traditional interpretations of tergal
origin. By comparing the muscle arrangement with the expression of leg patterning genes in Tribolium castaneum
embryos, we suggest that subcoxa-1 corresponds to the crustacean precoxa, and the molecular BTA, or the boundary
between the tergum and precoxa, is located more dorsally than the morphological BTA identified in C. insulicola.
Combining these morphological and molecular data, we propose the “precoxal theory” for the origin of insect wings.
Embryological evidence for the subcoxal theory
The subcoxal theory of insects postulates thatthe pleuron is derived from the proximal region of
the thoracic appendage, i.e., the subcoxa. The theory
was pioneered by the embryological observations of
Heymons (1899) in the hemipteran Naucoris cimicoides.
Although the theory was then advocated and developedby such authorities in insect morphology as Snodgrass(1927), Weber (1928, 1952), and Matsuda (1970), there
were few embryological studies (Roonwal, 1937). In
recent years, however, embryological evidence forthe theory has accumulated in Galloisiana yuasai(Grylloblattodea) (Uchifune and Machida, 2005),
Dineutus mellyi (Coleoptera) (Komatsu and Kobayashi,2012), Carabus insulicola (Coleoptera) (Kobayashi etal., 2013), Zorotypus caudelli (Zoraptera) (Mashimo
et al., 2014), and Gryllus bimaculatus (Orthoptera)(Mashimo and Machida, 2017). Kobayashi et al.
(2013) notably demonstrated that the subcoxa of C.
insulicola at about 35% developmental time (DT)becomes divided into a proximal subcoxa-1 and a
distal subcoxa-2, and as development proceeds, asemi-circular furrow, which corresponds to the future
paracoxal suture (PCXS), appears between the two
subcoxal regions. By comparing these two regions withthe spatial arrangement of larval thoracic pleurites,
Kobayashi et al. (2013) pointed out that the embryonicsubcoxa-1 and subcoxa-2 correspond to the larval

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anapleuron and katapleuron, respectively. Kobayashi(2017) continued this work by showing the presenceof subcoxae-1 and 2 in the embryos of D. mellyi,
Protohermes grandis (Megaloptera), and Nemotaulius
admorsus (Trichoptera). However, the subdivision of
the embryonic subcoxa has not yet been established in
non-holometabolous orders.
An alternative interpretation of the insect
subcoxa is offered by Mashimo and Machida (2017).

According to these authors, the thoracic pleuron of Gr.
bimaculatus completes by the rapid dorsal extension ofa single subcoxal region in late embryonic stages. Theyinterpreted the subcoxa of Ga. yuasai and the subcoxa-1
of C. insulicola as part of the tergum, and thus the
PCXS of the latter is not an internal structure of thesubcoxa (i.e., a subdivision within the appendage) but
the boundary between the tergum and the appendage(BTA).

Fig. 1 Lateral views of the larva of Carabus insulicola, fixed with alcoholic Bouinʼs fluid. A. Newly hatched larva. The cuticle is
not yet sclerotized, so some muscles can be observed through the semi-transparent cuticle. The regions derived from theembryonic subcoxae-1 and 2 are highlighted in blue and red, respectively. B. About two days after hatching. A-Aʼ, B-Bʼ: levelsof cross sections in Fig. 3A, B, respectively. cx: coxa, hc: head capsule, mrBTA: morphological boundary between tergum andappendage, PCXS: paracoxal suture, pnl: paranotal lobe, saf: subalar fold, scx-1, 2: subcoxa-1, 2, sp: spiracle, th1-3.tg: pro-,meso-, and metathoracic tergum, tr: trochanter, arrowhead: long seta near mrBTA.

PARANOTAL LOBES ARE APPENDICULAR IN ORIGIN

Micro-CT analysis of the muscular system in the
Carabus insulicola larva
To resolve the disagreement over the PCXS, westarted with the fact that podomeres (leg segments)
are defined as having joints on which muscles either
originate or insert. We reasoned that, if subcoxae-1
and 2 are indeed derived from ancestral podomeres,
then the ancestral joint between them (our PXCS)
should have muscles originating or inserting on it. To
determine whether the PCXS has muscles originating
or inserting on it, we examined the thoracic muscularsystem of newly hatched C. insulicola larvae using
an X-ray micro-CT scanner (XRadia Micro XCT400 system, ZEISS). Here, we identify all meso- andmetathoracic muscles according to Matsudaʼs (1970)
system. Prothoracic muscles were excluded from theexamination because the deformation of the segment
would deter identification of musculature.
Subcoxae-1 and 2 are independent podomeres
In insects, the PCXS (Fig. 1A) forms an internal
paracoxal ridge PCXR (Figs. 2, 6, PCXR) on which
several transverse muscles attach (e.g., Matsuda, 1970;
Vilhelmsen, 2000; Willkommen, 2008; Wipfler etal., 2015). We compared our data to previous studiesof the muscular system of other insects, including
representatives from Ephemeroptera, Plecoptera,

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Orthoptera, and Odonata (e.g., Snodgrass, 1935,
Matsuda, 1970, Willkommen, 2008). However, the mostdetailed and precise description of muscles attaching
to the PCXR has been provided only by Willkommen(2008) in Ephemeroptera. In her paper, muscles t-p 3
and p-cx 5 sensu Matsuda are attached to the dorsal sideand ventral side of the anterior PCXR, respectively (cf.
Willkommen, 2008; Figs. 16, 17, muscle AN.Pm = t-p 3,
P.Cm = p-cx 5). These two muscles specifically attachedto the PCXR are observed in the exact same positions
in C. insulicola (Fig. 2). Therefore, the PCXS/PCXR
is not the BTA, hence the division of the subcoxa into
subcoxae-1 and 2 is valid (Figs. 1A, 3A, PCXS).
As postulated by Weber (1952), the furca should
be regarded as a specialized apodeme formed in the
medial part of the PCXS (Fig. 4, fup). In fact, in Carabus
larvae, a furcal apodeme is located in the sternum
slightly distant from the medio-posterior margin of thecoxal base of each thoracic leg (Zaitsev, 2008). Manymuscles are observed to be attached to the furca in thelarvae of C. insulicola (Fig. 3A, B, circles indicated bywhite dotted lines), providing further evidence that
the PCXS is the remnant of the ancestral joint between
subcoxae-1 and 2.
To determine whether subcoxa-1 (anapleuron)and subcoxa-2 (katapleuron) are derived from two
independent podomeres, we analyzed the intrinsic

Fig. 2 Parasagittal-sectioned micro-CT image of the newly hatched larva of Carabus insulicola, mesal view of the right half ofpterothoraxes. The larva in this image is the same as shown in Fig.1A. The image of the alimentary canal was digitally removed
from the original image. Abbreviations for muscles follow the Matsudaʼs system and are highlighted in yellow letters. Red
circles indicate intrinsic muscles of subcoxa-1. Blue, green, and white circles indicate pleuro-coxal muscles, “furca-coxal”
muscles, and a “pleuro-furcal” muscle of subcoxa-2, respectively. cx: coxa, PCXR: paracoxal ridge, sp: spiracle, th1, 2.tg; proand mesothoracic tergum.

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Fig. 3 Cross-sectioned micro-CT images of the posterior part of the metathorax of Carabus insulicola at the levels of A-Aʼ (A) and
B-Bʼ (B) in Fig. 1A. The larva in this image is the same as in Fig.1A. The image of the alimentary canal was digitally removed
from the original image. Abbreviations for muscles follow the Matsudaʼs system and are highlighted in yellow letters. Red
circles indicate intrinsic muscles of subcoxa-1 (precoxa). Blue, green, and white circles indicate pleuro-coxal muscles, “furcacoxal” muscles, and a “pleuro-furcal” muscle, respectively, of subcoxa 2. Circles indicated by white dotted lines show theplaces where a furcal invagination is formed in the larva with sclerotization. ab1.g: first abdominal ganglion, cx: coxa, mlBTA:
molecular boundary between tergum and appendage inferred from gene expression data of Tribolium castaneum embryos,
mrBTA: morphological boundary between tergum and appendage, PCXS: paracoxal suture, saf: subalar fold, scx-1, 2:
subcoxae-1 and 2, scx-2-cx j: subcoxa-2-coxa joint, th3.tg: metathoracic tergum, tr: trochanter.

PARANOTAL LOBES ARE APPENDICULAR IN ORIGIN

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muscles of each region. In subcoxa-2, six muscles
were found to have both their origin and insertion
within this region (Figs. 2, 3A, B, 4); that is, three ofthem were pleuro-coxal muscles (p-cx 5, p-cx 6, andt-cx 8 sensu Matsuda), two were newly defined “furcacoxal” muscles (s-cx 2 and s-cx 6 sensu Matsuda), andthe other one was the newly defined “pleuro-furcal”
muscle (p-s 1 sensu Matsuda). Given that muscles
originate or attach on both the proximal and distal“joints” of subcoxa-2, this region (katapleuron) should
be regarded as an independent podomere.
In subcoxa-1, two muscles (t-p 16 and t-s 4 sensu
Matsuda in Figs. 2 and 3A, B) are localized and bothare attached to the subalar fold (Figs. 3A, 4, saf). The
subalar fold is observed from the outside as a faintsuture in subcoxa-1 running from its medio-ventral
part to its dorsoposterior end (Fig. 1A, B, saf). This foldis first documented in the larvae of the megalopteran
Corydalus cornutus, and muscle 161 (epimeral-subalar
muscle) (t-p 16 sensu Matsuda) is attached to thisfold (Kelsey, 1957). In the adult of this species, thesubalare is formed just dorsal to this fold. Muscle t-s 4
has been known from only Ephemeroptera (in both
adults and larvae) as the subalar-furcal muscle (SA.
Fm sensu Willkommen, 2008). Our observation ofthis muscle in C. insulicola is therefore noteworthy.
Although Matsuda (1970) regarded the subalare as atergal structure, Snodgrass (1935) and Willkommen(2009) regarded it as belonging to the pleuron. Basedon our analysis, muscle t-p 14 originates from near thelateral edge of the tergum (Fig. 3A) and inserts in theantero-dorsal part of the subalar fold (not pictured
in Fig. 3A). In Ephemeroptera, the superior axillarpleural muscle (Ax.PmS sensu Willkommen, 2008)
runs from the third wing base sclerite to the anterior
part of the subalar. In the adult of Carabus, t-p 14 also
runs from the third axillary sclerite to the pleuron(epimeron) (Larsén, 1966), although, in Carabus
as in other Neoptera, the anterior subalar is absent(only posterior one is present). Willkommen (2008),
thus, concluded t-p 14 in Neoptera and Ax.PmS in
Ephemeroptera are homologous muscles. Therefore,
the arrangement of t-p 14 also suggests that the subalar
is indeed a pleural structure. This conclusion agrees
with the recent findings from Ohde et. al. (2022),
where ablation of the pleuron in 3rd instar nymphsof the cricket Gr. bimaculatus results in the loss of the
epipleurites including the basalare and subalare. We
therefore agree with the interpretation of Snodgrass andWillkommen. Subcoxa-1 (anapleuron), therefore, canbe regarded as an independent podomere that would
be homologous to the crustacean precoxa (the mostproximal podomere). The existence of the precoxa in
Crustacea has been demonstrated in the study of postembryological development of decapods by Fukuda(1994) and also in the study of gene expression in
amphipods by Bruce and Patel (2020) and Bruce (2021).
The PCXS and PCXR, thus, should be newly interpreted
as the segmental boundary between the precoxa andsubcoxa.

Fig. 4 Intrinsic muscles of subcoxae-1 and 2 mapped on theventral view of a schematic thoracic segment (modified
from Weber, 1952, Fig. 16c). Intrinsic muscles ofsubcoxa-1 (precoxa) are highlighted in red. Intrinsic
muscles of subcoxa-2, i.e., pleuro-coxal muscles, arehighlighted in blue, furca-coxal muscles in green, andpleuro-furcal muscle in orange. Abbreviations formuscles follow Matsuda (1970). fup: furcal pit, pls:
pleural suture, PXCS: paracoxal suture, saf: subalar fold,scx-1, 2: subcoxae-1 and 2, tg: tergum.

Paranotal lobe is derived from the dorsal half ofsubcoxa-1 (precoxa)
Snodgrass (1935) defined a morphologicalboundary between the “dorsum” and “appendage”(subcoxa), which appears to correspond to the BTA ofMashimo and Machida (2017). Snodgrassʼ “dorsum”,
however, includes laterotergites on both sides of themedian “principal tergum” (Snodgrass, 1935, Fig.
139). Based on the muscular system described here,
we conclude that his laterotergite corresponds tothe ventral half of our subcoxa-1 (precoxa) and his“subcoxa” to our subcoxa-2. Snodgrassʼ definition thus
cannot be applied to our morphological boundary
between the tergum and appendage (mrBTA) described
below.
In the newly hatched larvae of C. insulicola,
the mrBTA can be seen as a faint horizontal suture(Fig. 1A). In the larvae after sclerotization, a highly
sclerotized ridge is observed in the same place where
the suture was present (Fig. 1B). A long seta is present
near the posterior end of this suture or sclerotized ridge
as a landmark of the mrBTA in each thoracic segment(Fig. 1A, B, arrowhead). Within a day after hatching thedorsal half of the larval precoxa (subcoxa-1) slightly

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extends laterally, accompanied by sclerotization, andforms an eave-like structure, or the paranotal lobe (Figs.
1B, 6, pnl), whereas the ventral half forms the pleuron. It
is noteworthy that muscle t-p 14, or the axillary muscle,
is attached to the tergum just dorsal to the mrBTA (Fig.
3A, t-p 14). As mentioned before, in the adult, thismuscle is assumed to be inserted on the third axillarysclerite of the wing base (Larsén, 1966; Matsuda, 1970).
Judging from the position of the mrBTA, therefore,axillary sclerites of the wing base are assumed to be in
the tergum, but not in the paranotal lobe.
In comparison of our mrBTA to the BTA ofMashimo and Machida (2017), our mrBTA would
correspond to their “paratergal groove” (e.g., their
Figure 2F, G, white arrows), and our subcoxa-1 to their“paratergal bulge” from which a wing develops later.
The BTA of Mashimo and Machida (2017) would thus
correspond to the boundary between subcoxae-1 and 2,
or our PCXS.
Incongruence between morphological and molecularboundary between tergum and appendage
According to Bruce and Patel (2020), theparanotal lobe is homologous to the insect wing andthe crustacean tergal plate, both of which are thelateral expansions of the precoxa, the proximal-mostFig. 5 Expression of pannier, aurucan, and serrate in Tribolium
castaneum embryo at the stage of leg segmentation,
ventrolateral view. A. pannier (pnr) in red marks thedorsal-most tissue, or the bona fide tergum. araucan(ara) in green brackets subcoxa-1 and is also expressedin a patch on the coxa. serrate (ser) in blue marks thelocation of joints, as well as the presumptive wing
(Couso et al., 1995). B. Gene expression highlighted in
the mesothoracic and the first abdominal segment. C.
Embryo without gene expression. ab1: first abdominal
segment, b.f.tg: bona fide tergum, cx: coxa, cx-tr j: coxatrochanter joint, DAPI: DAPI staining, fm: femur, fmtbtar j: femur-tibiotarsus joint, hl: head lobe, lb: labium,
mlBTA: molecular boundary between tergum andappendage, mx: maxilla, mxp: maxillary palp, PCXS:
paracoxal suture, pp: pleuropodium, ptar: pretarsus,
p.wg: presumptive wing, scx-1, 2: subcoxae-1 and 2,
scx-2-cx j: subcoxa-2-coxa joint, sp: spiracle, tbtar:
tibiotarsus, tbtar-ptar j: tibiotarsus-pretarsus joint, th1,
3.l: prothoracic- and metathoracic leg, tr: trochanter, trfm j: trochanter-femur joint.

Fig. 6. Schematic diagram showing regionally categorized
integuments of the pterothorax in the Carabus insulicola
larva, cross sectioned. The regions of bona fide tergum(b.f.tg), precoxa-derived tergum (pcx.d.tg), and thelocation of the molecular BTA (mlBTA) are inferredfrom gene expression data of Tribolium castaneum
embryos. bs: basisternum, cx: coxa, fs: furcasternum,
fur: furca, mrBTA: morphological BTA, pcx: precoxa,
PCXR: paracoxal ridge, PCXS: paracoxal suture, pnl:
paranotal lobe, scx-1, 2: subcoxae-1 and 2.

PARANOTAL LOBES ARE APPENDICULAR IN ORIGIN

podomere. If the paranotal lobe is the lateral expansionof a podomere, then the real BTA must be dorsal tothe paranotal lobe. This hypothesis is supported by thefollowing gene expression data.
Bruce and Patel (2020) and Bruce (2021)
concluded that the bona fide tergum (true body wall)in all arthropods is marked by pannier, while araucan
expression brackets the precoxa (subcoxa-1) dorsally– where the precoxa (subcoxa-1) meets the bonafide tergum – and ventrally – at the joint between
subcoxae-1 and 2. Additionally, previous studies havefound that serrate mediates the formation of leg joints(de Celis et a., 1998). Thus, the molecular BTA (mlBTA),
or the boundary between the bona fide tergum andprecoxa, should be located within the overlap between
pannier and the dorsal domain of serrate. Furthermore,
if subcoxae-1 and 2 are true podomeres, serrate should
be expressed in each of their proximal and distal joints.
Notably, the expression of these genes provides three
separate indicators of the location of the mlBTA.
We examined the expression of the above three
genes in Tribolium castaneum embryos. As predicted,
pannier is expressed in the dorsal-most tissue, the bonafide tergum; araucan expression brackets subcoxa-1;
and serrate is expressed in all joints, including theproximal and distal joints of both subcoxae-1 and
2 (Fig. 5A, B). Therefore, subcoxae-1 and 2 are true
podomeres. Notably, the molecular location of the
BTA (Fig. 5A, mlBTA) – the region of overlap between
pannier and serrate – occurs dorsal to the thickhorizontal domain of serrate expression. This serrate
domain partially overlaps with the presumptive wing
(Fig. 5A, B, p.wg), marked by vestigial expression (Bruceand Patel, 2020, Fig. 3l, extended data, Fig. 8b).
Another notable observation when considering
the data in Bruce and Patel 2020 is that, like the BTA,
the PCXS also has a molecular signature; that is, theventral armband of araucan expression together witha serrate joint domain (Fig. 5A, B, PCXS). In insects,
this marks the boundary between subcoxae-1 and 2,
while in crustaceans it marks the boundary between
the precoxa and the crustacean coxa. Thus, the BTA
and PCXS have distinct molecular signatures, which
can be applied to and investigated in other insect
systems. Examination of pannier, araucan, and serrate
expression in Gr. bimaculatus embryos, for example
using the inexpensive and forgiving in situ HCR v3.0
(Choi et al., 2018; Bruce et al., 2021), may resolve thedifferent interpretations of Kobayashi et al. (2013) and
Mashimo and Machida (2017).
It is interesting to note the somewhat unexpectedshape of serrate expression around the perimeter ofsubcoxa-1. This unexpected shape makes sense when
one considers that a) serrate is expressed in a perimeteraround each leg joint, and b) subcoxa-1 is a flattened

7cylinder rather than an elongate cylinder like the distal
leg segments. Thus, when the embryo is in ventrolateralview (Fig. 5C), the joint between subcoxa-1 andthe bona fide tergum is viewed at an angle of about
60-degree, and appears more circular (Fig. 5A, B,
mlBTA), while the distal leg joints are viewed edge-on,
and appear like a line (Fig. 5B, scx-2-cx j, cx-tr j, tr-fm j,
fm-tbtar j).
“Precoxal theory” for the origin of insect wings

Our musculature and gene expression datastrongly suggest the positional incongruence between
the morphological and molecular BTAs; that is, thelatter is located more dorsally than the former (Fig.
6). We believe the mlBTA lies along the lateral edgeof the budged area of the median tergum (Figs. 3A, 6,
mlBTA). The incongruence is not necessarily unnatural,
because sclerites do not always define anatomical areas(Snodgrass, 1935). However, if the molecular BTA
but not the morphological BTA corresponds to a real
BTA, the incongruence implies that the lateral part ofthe so-called tergum is really derived from the precoxa(precoxa-derived secondary tergum) (Fig. 6, pcx.d.tg)
and only the medial part (bona fide tergum) (Fig. 6,
b.f.tg) should be regarded as a primary tergum.
Based on the mlBTA, therefore, the tergo-sternal(furcal) (t-s 3) and tergo-pleural (t-p 14) muscles (Figs.
2, 3A), which are attached to the first and third axillarysclerites in the adult, must be included in the precoxal
intrinsic muscles. Moreover, more medial muscles suchas the tergo-pleural (t-p 15) and tergo-coxal (t-cx 3 to7) muscles (Figs. 2, 3A) also must be categorized into a
precoxal intrinsic muscle and precoxal-coxal muscles,
respectively. Underneath the bona fide tergum, i.e.,
between the right and left mlBTAs, there exists only thebroad tergal muscles consisting of t 11 to 14, and noneof the dorso-ventral muscles are in this region (Fig. 3A).
The detail of these changes in muscle category will be
discussed in our subsequent paper.
The origin of insect wings has long been debated.
One theory holds that wings derive from paranotal
lobes (edges of the tergum) (Crampton, 1916), while
another theory is that wings emerge from an ancestralproximal leg segment that now functions as thedorsal half of the pleuron (Kukalová-Peck, 1983,2008). Recently, it has been proposed that both ofthese theories are correct, but in different ways. The“dual origin” theory of Clark-Hachtel and Tomoyasu(2020) proposes that wings represent a composite ofboth tergal and appendicular elements. In contrast,
Bruce and Patel (2020) proposed that both the exite
and paranotal theories are correct, but each pertains
to different phylogenetic timepoints: the crustaceanprecoxal exite evolved into paranotal lobes, which
later evolved into insect wings, and therefore wings

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Y. KOBAYASHI ET AL.

are derived only from the precoxa. In support of thelatter interpretation, Ohde et. al. (2022) found that thepleuron does not contribute to the wing, but instead
forms part of the epipleurites, such as the basalare andsubalare.
Combining the positional information of the
mrBTA and mlBTA obtained here, our data supports
the growing consensus that paranotal lobes and insectwings evolved from the exite of the precoxa, and thatthe precoxa now forms a secondary tergum. We callthis the precoxal theory for the origin of insect wings.
We note that our precoxal theory is similar to KukalováPeckʼs epicoxal theory, with the exception that ourprecoxa is more expansive, encompassing all elementsof wing articulation but also the lateral tergum and partof the sternum (basisternum). Our precoxal theory
should be verified in many insect taxa in the future.
Acknowledgements: We are grateful to Prof.
N. H. Patel (Marine Biological Laboratory, Woods
Hole, USA) for his kindness in providing us reagentsand space for the gene expression experiments. We
also thank two anonymous referees for their criticalcomments that helped to improve the manuscript.
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