INTERSEXUALITY IN THE BURROWING CRAYFISH, PARASTACUS

PILIMANUS (VON MARTENS, 1869) (DECAPODA, PARASTACIDAE)

BY

ERICH H. RUDOLPH1,3 ) and ANA C. VERDI2 )

1 ) Departamento de Ciencias Básicas, Universidad de Los Lagos, Casilla 933, Osorno, Chile
2 ) Sección Entomología, Facultad de Ciencias, Universidad de la República, P.O. Box 10773,
Montevideo, Uruguay

ABSTRACT

Various authors have reported the occurrence of supernumerary gonopores in many species of
Parastacus, which has generated discussion about their sexual system. In this study, we examine
the external and internal sexual characters of 102 specimens of the burrowing crayfish, Parastacus
pilimanus, captured in the province of Maldonado in southeast Uruguay, with the aim of clarifying its
sexual system. In this study, we show that in all the specimens analysed, female and male gonopores
co-existed externally, and we verified the presence of only one type of gonad internally: either
ovary (intersex females) or testis (intersex males), from which one pair of oviducts and another
of sperm ducts originated, connecting the gonad to the respective gonopores. We also verified that
all specimens smaller than 27.2 mm cephalothorax length (CL) (N = 73) had the same external
morphology. Nevertheless, dissection revealed that their gonads were already differentiated. We
established that 54.8% of these specimens were intersex females, while the remaining percentage
corresponded to intersex males. Twenty-nine specimens were found with a CL equal to, or above,
27.2 mm. Of these, 34.5% (N = 10) were intersex males with the same external morphological traits
that characterize all specimens smaller than 27.2 mm CL. The remaining 19 specimens (65.5%) were
intersex females, 15 of which presented secondary sexual characters, probably associated with egg
incubation. The evidence gathered suggests that Parastacus pilimanus has permanent intersexuality,
similar to Parastacus pugnax and Parastacus varicosus in Uruguay. We conclude by identifying
some topics that require further study, in order to understand the adaptive value of this type of
intersexuality.

RESUMEN

Varios autores han reportado la ocurrencia de gonoporos supernumerarios en muchas especies

de Parastacus, lo que ha generado discusión acerca del sistema sexual de ellas. En este trabajo
nosotros examinamos los caracteres sexuales externos e internos de 102 especimenes del camarón
excavador Parastacus pilimanus capturados en el Departamento de Maldonado, sureste de Uruguay,

3 ) Fax: +56·64333455; e-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2010 Crustaceana 83 (1): 73-87

Also available online: www.brill.nl/cr DOI:10.1163/001121609X12530988607551
74 ERICH H. RUDOLPH & ANA C. VERDI

con el objetivo de clarificar su sistema sexual. Aquí nosotros demostramos que todos los espec-
imenes analizados presentaron externamente coexistencia de gonoporos de hembra y de macho, e
internamente una gónada única — ya sea ovario (hembras intersexo) o testículo (machos intersexo)
— desde la cual se originan un par de oviductos y otro de espermiductos que la conectan a los respec-
tivos gonoporos. También comprobamos que todos los especimenes de tallas menores a 27,2 mm de
longitud cefalotorácica (LCT) (N = 73), tienen la misma morfología externa. No obstante, las disec-
ciones demostraron que sus gónadas ya estaban diferenciadas. En ellos comprobamos que el 54,8%
eran hembras intersexo y el porcentaje restante machos intersexo. De tallas iguales o superiores a
27,2 mm de LCT, encontramos 29 especimenes. De ellos, el 34,5% (N = 10) eran machos intersexo
los que presentaron los mismos rasgos morfológicos externos que caracterizan a todos los espec-
imenes de tallas inferiores a 27,2 mm de LCT. Los 19 especimenes restantes (65,5%) eran hembras
intersexo, en 15 de las cuales comprobamos la presencia de caracteres sexuales secundarios, proba-
blemente asociados a la incubación de los huevos. Las evidencias reunidas sugieren que Parastacus
pilimanus, al igual que Parastacus pugnax y Parastacus varicosus de Uruguay, presenta intersexuali-
dad permanente. Nosotros concluimos sugiriendo algunos tópicos que necesitan de mayores estudios
para comprender el valor adaptativo de este tipo de intersexualidad.

INTRODUCTION

Intersexuality is characterized by the occurrence of sexual characters of both

sexes in the same individual. It can be limited to the external morphology, or
extend to gonadal differentiation, and can be present in both gonochoristic and
hermaphroditic species (Khalaila & Sagi, 1997). Its origin is attributed to different
causes, e.g., parasitary or viral infections (Ginsburger-Vogel, 1991; Juchault et al.,
1991), genetic or hormonal anomalies (Zou & Fingerman, 2000; Parnes et al.,
2003; Weeks et al., 2006), and to the effects of contaminating chemicals present in
the environment (Ford et al., 2004; Jungmann et al., 2004). Over the last few years,
numerous cases of intersexuality have been reported in Malacostraca, especially in
Amphipoda (cf. Dunn et al., 1994; Ladewig et al., 2003; McCurdy et al., 2004;
Ford & Fernandes, 2005), Isopoda (cf. Korczynski, 1988; Rigaud & Juchault,
1998), and Decapoda (cf. Galil & Tom, 1990; Laurent, 1991; Micheli, 1991;
Zou & Fingerman, 2000; Hanamura & Ohtsuka, 2003; Turra, 2004; Tang et al.,
2005). In freshwater crayfishes of the family Parastacidae, intersexed species have
been reported in the genera Engaeus, Engaewa, Cherax, Euastacus, Parastacus,
Samastacus, and Virilastacus (cf. Horwitz, 1988; Medley & Rouse, 1993; Honan
& Mitchell, 1995; Rudolph & Almeida, 2000; Rudolph et al., 2007).
Ever since Von Martens (1869) and Faxon (1898) established that Parastacus
species are characterized by the occurrence of supernumerary gonopores, their
sexuality has been the subject of discussion. The functional significance of the
supernumerary gonopores in this genus has been poorly documented for many
years, mainly due to the small number of specimens analysed (Von Ihéring, 1893;
Lönnberg, 1898; Hay, 1905; Turner, 1935; Thompson, 1982). While Lönnberg
 INTERSEXUALITY IN PARASTACUS PILIMANUS (VON MARTENS) 75

(1898) and Hay (1905) postulated a probable hermaphroditism, the other authors
cited discarded that alternative. Nevertheless, from the 1990s onwards, studies on
the sexual system of this species have notably increased. Evidence gathered in
these recent studies permits to distinguish two sexual systems in Parastacus: partial
protandric hermaphroditism in Parastacus nicoleti (Philippi, 1882), Parastacus
brasiliensis (Von Martens, 1869), and Parastacus defossus Faxon, 1898 (cf.
Rudolph, 1995; Almeida & Buckup, 2000; Noro et al., 2008), and permanent
intersexuality in Parastacus pugnax (Poeppig, 1835) (cf. Rudolph, 1997). The
sexual system of Parastacus varicosus Faxon, 1898, has been studied by Rudolph
et al. (2001) and by Silva-Castiglioni et al. (2008) in Uruguayan and Brazilian
populations, respectively. Nevertheless, this species can not be classified into
any of the aforementioned sexual systems, because the results obtained are
contradictory. According to Rudolph et al. (2001), the species presents permanent
intersexuality, whereas Silva-Castiglioni et al. (2008) classify it as a protandric
hermaphrodite.
Parastacus pilimanus (Von Martens, 1869) is a burrowing species that con-
structs shallow galleries (maximum 30 cm) with numerous entrances (3 to 7), that
converge in a large central chamber (Buckup & Rossi, 1980). Its geographic dis-
tribution includes the southeast of the Río Grande do Sul state in Brazil, northeast
Argentina, and southeast Uruguay (Amestoy, 1982; Morrone & Lopretto, 1994).
Only some aspects of the biology of this species are known, related to its burrow-
ing behaviour (Amestoy, 1982) and early embryonic and postembryonic develop-
ment under laboratory conditions (Amestoy, 1983). Thompson (1982) analysed the
anatomy and gonadal histology of 100 Parastacus pilimanus and Parastacus vari-
cosus females, concluding that they have gonopores of both sexes, connected to the
ovary through a double set of gonoducts. From then on, no further contributions
have been made to the knowledge on the sexuality of Parastacus pilimanus.
This article describes the external and internal sexual characters of Parastacus
pilimanus, aimed at clarifying the sexual system of this South American parastacid.

MATERIAL AND METHODS

One-hundred-and-two specimens of Parastacus pilimanus were collected in

July 1998, using a partial vacuum pump, in the province of Maldonado
(34◦ 54 09 S 54◦ 56 55 W), in southeast Uruguay. Immediately after capture, the
specimens were fixed in Bouin’s solution for 24 hours and then conserved in 70%
ethanol. In the laboratory, the descriptions and keys of Riek (1971) and Hobbs
(1974) were used to identify the species. Morphological characteristics of each
specimen were examined under a stereomicroscope, equipped with a drawing
76 ERICH H. RUDOLPH & ANA C. VERDI

chamber. The morphometric data, recorded using callipers to 0.01 mm accuracy,

were: cephalothorax length (CL), length of abdominal somites, width of abdom-
inal somites 2 to 6, height of the pleura on abdominal somites 2 to 5, length of
the pleuron on abdominal somite 2, length and width of the telson, and length
and width of the uropodal endopodite and exopodite. The relationship between the
cephalothorax length and each of these morphometric characters was verified, in
the male and female intersexes, through regression analysis. In each regression a
Student’s t-test was applied, to establish differences between the respective slopes
(Zar, 1999). The sex ratio was compared with a hypothetical 1 : 1 relationship, us-
ing the chi-square test with Yates correction. Width of the gonoducts was measured
using an ocular micrometer. Some specimens (N = 78) of different sizes were also
selected to analyse gonad and gonoduct anatomy. Subsequently, these structures
were removed for histological study that began with dehydration in a series of
alcohols, followed by diaphanization and subsequent embedding in paraffin. Sec-
tions, 7 μm thick, were stained with hematoxylin-eosin. In order to identify and
characterize gonadal histology, the following descriptions and photomicrographs
were used: Hobbs et al. (2007) in Astacidea, Payen (1973) in Astacus leptodactylus
(Eschscholtz, 1823), Rudolph (1995, 1997) in Parastacus nicoleti and Parastacus
pugnax, respectively, and Rudolph et al. (2001) in Parastacus varicosus. The ter-
minology proposed by Hobbs et al. (2007) was adopted to designate the structures
of the male genitalia. A Wild equipment was used for photomicrographic record-
ing of the external and internal sexual characters. Finally, the photographs were
manipulated in Adobe Photoshop 6.0.

RESULTS

External sexual characters

Coexistence of female and male gonopores was observed in all specimens of

Parastacus pilimanus collected (fig. 1). Analysis of the morphological characteris-
tics of their gonopores disclosed two sexual forms:
Intersex 1. — Specimens (N = 87) with female gonopores, internal border more
prominent than external border, giving them a semi-ellipsoidal aspect, in the coxae
of the third pair of pereiopods (P3 ). No setae were observed on either of these
borders, or close to them. The cuticle that covered them was slightly convex,
calcified, with no trace of an opening (fig. 2A). These specimens also showed a
small, fixed, calcified phallic papilla, close to the internal border of the ventral
surface of the coxae of the fifth pair of pereiopods (P5 ) in the form of a truncated
cone, and a male gonopore that opens onto its apical end. Both P5 coxae show male
 INTERSEXUALITY IN PARASTACUS PILIMANUS (VON MARTENS) 77

Fig. 1. Parastacus pilimanus (Von Martens, 1869). Sternal region and coxae of pereiopods 2 through
5, showing the coexistence of male and female gonopores. FG, female gonopore; PP, phallic papillae;
Co, coxa; Ba, basis; P2 , second pair of pereiopods; P5 , fifth pair of pereiopods. Scale bar = 2.0 mm.

cuticle partition (fig. 2B). The size of these intersex specimens ranged from 10.8
to 44.5 mm CL.
Intersex 2.— Specimens (N = 15) with ellipsoidal female gonopores, external
borders as prominent as internal borders. With many, relatively long setae on the
surface of the coxa close to its external borders. Furthermore, these gonopores
were covered with a non-calcified, semi-transparent, slightly convex membrane,
or, alternatively, partially calcified, flat, and non-transparent. In both cases, these
membranes presented a semi-ellipsoidal opening in the zone closest to the internal
border of the gonopores (fig. 2C). The phallic papillae were morphologically
similar to the papillae described in intersex 1. However, unlike the latter, they
presented many setae around the base of the cone (fig. 2D). Both P5 coxae also
showed a male cuticle partition. The size of these intersexes ranged from 27.2 to
45.3 mm CL.

Anatomy of gonads and gonoducts

The gonad of Parastacus pilimanus is formed by a pair of longitudinal lobes,
in close proximity to each other in the intersex females, with oocytes in advanced
vitellogenesis and slightly more separated in the intersex males and females, with
ovaries in previtellogenesis or incipient vitellogenesis. Nevertheless, no connecting
transversal commissure was observed. Each of these lobes comprised an anterior
 78
ERICH H. RUDOLPH & ANA C. VERDI

Fig. 2. Parastacus pilimanus (Von Martens, 1869). Gonopore morphology of the intersexes 1 and 2. A, B, female and male gonopores of intersexes 1,
respectively; C, D, female and male gonopores of intersexes 2, respectively; Co3, coxopodite of P3 ; Se, setae; FG, female gonopore; Co5, coxopodite of
P5 ; PP, phallic papillae; GA, genital atrium; MCP, male cuticle partition. Scale bars = 1.0 mm.
 INTERSEXUALITY IN PARASTACUS PILIMANUS (VON MARTENS) 79

zone, dorsoventrally adjacent to the anterior hepatopancreas, and another, larger,

posterior zone, located under the heart, that extends posterolaterally over the
posterior hepatopancreas and/or the flexor and transverse thoracic muscles, without
invading the abdomen. The gonoducts of both sexes originate from this last portion.
The oviducts emerge close to the base of the anterior regions of the gonadic lobes.
From there, they descend directly towards the P3 coxae. Furthermore, they were
flat, of a similar width all along, and without a seminal receptacle. The sperm ducts
originated from the middle part of the posterior portion of each gonadic lobe, and
from there they ran towards the P5 coxae. All specimens presented gonoducts of
both sexes, that connected one entirely female or entirely male gonad with the
respective gonopores.
When the gonad was a testis, the oviducts were thin and translucent (=
0.27 mm; SD = ±0.057; N = 12). In contrast, the sperm ducts were then
cylindrical and it was possible to distinguish a short, straight, thin proximal
portion; another wider medial portion (= 0.75 mm; SD = ±0.122; N = 12),
curved ventrally; and finally a distal portion, thinner (= 0.39 mm; ±0.151; N =
12) but longer than the anterior part. This distal part penetrated the musculature of
the P5 coxae and was observed to be connected to the ejaculatory zone of the sperm
duct (fig. 3A). This latter opens to the exterior through the genital atrium, located
at the end of a small, truncated, cone-shaped phallic papilla. In larger specimens,
it was possible to distinguish the androgenic gland adjacent to the terminal third of
the sperm duct’s distal portion.
When the gonad was an ovary, the oviducts were wide (= 0.63 mm; SD =
±0.15; N = 11). The sperm ducts, on the other hand, then were thin (= 0.29 mm;
SD = ±0.014; N = 11) and flat. Due to the aforementioned, it was not possible to
distinguish the various sections observed in the sperm ducts of the specimens with
testes (fig. 3B). Neither was it possible to distinguish the androgenic gland in this
type of sperm duct.
All specimens smaller than 27.2 mm CL (N = 73) had supernumerary gono-
pores with the morphological characteristics described for intersex 1. Neverthe-
less, once dissected, it was observed that the gonad of each of these specimens was
either an ovary, or a testis. Thus, it was established that 40 individuals (54.8%)
were intersex females and 33 (45.2%), intersex males. Twenty-nine specimens
were found to be of sizes equal to or larger than 27.2 mm CL. According to the
morphology of the female gonopores, it was found that 14 of these latter specimens
were intersex 1 and the remaining 15 were intersex 2. However, dissection of these
29 specimens revealed that 19 (65.5%) were intersex females, because their gonads
were an ovary, and the remaining 10 (34.4%), whose gonads were a testis, intersex
males. This showed that at sizes equal to or above 27.2 mm CL, 28.6% (N = 4)
of the intersex 1 specimens were intersex females, whose external morphology did
not yet exhibit the female condition.
80 ERICH H. RUDOLPH & ANA C. VERDI

Gonadal histology

Independent of the size of the specimens analysed, the testes of Parastacus pili-
manus were whitish. They consisted of testicular acini and collecting tubules, sur-
rounded by connective tissue. Each acinus contained cells in a similar spermato-
genetic stage, thus some were observed with spermatogonia, others with sperma-
tocytes, or spermatids, or spermatozoa. The lumen of the collecting tubules can
either be empty or be filled with varying quantities of spermatozoa (fig. 3C).
The size and colour of Parastacus pilimanus ovaries not only depended on
the size of the females, but also on the vitellogenetic stage of the oocytes.
However, in general, in small, intersex females (<27.2 mm CL) the oocytes were
in previtellogenesis, thus both the oocytes and the ovaries were small and whitish.
In intersex females larger than 27.2 mm CL, oocytes were in primary or secondary
vitellogenesis; consequently, both the oocytes and the ovaries were larger and their
colour fluctuated between yellow and orange. The ovaries were surrounded by an
external muscular sheath. Oocytes surrounded by follicular cells were observed in
their interior, immersed in the ovarian stroma (fig. 3D). Coexistence of the ovarian
and testicular tissue in the same gonad was not observed in any of the histological
sections.

Sex ratio

After dissecting the 102 specimens captured, 43 were established to be intersex

males and 59 were intersex females, representing a sex ratio of 0.7 : 1 in favour
of females. This value did not differ significantly from a 1 : 1 sex ratio (χ 2 =
3.84, P > 0.05). When analysing sex ratio per size class, it was found that all
classes contained a larger number of females (table I). Under 29.9 mm CL, there
were 33 intersex males and 40 intersex females (0.8 : 1), above 29.9 mm, we found
10 intersex males and 19 intersex females (0.5 : 1).

Secondary sexual characters

No significant differences were found between the abdomens of intersex males

and females smaller than 27.2 mm CL. However, when comparing the abdomens
of the 15 intersex 2 females with those of the 10 intersex 1 males measuring over
27.2 mm in cephalothoracic length, significant differences (t0.05(2),21 = 2.080; P 
0.05) were found in 15 out of the 16 pairs of slopes analysed (table II). These
females had significantly broader abdomens and higher pleura than the male
intersex abdomens. Furthermore, the telson was wider and the uropodal endo-and
exopodites were longer and wider.
 INTERSEXUALITY IN PARASTACUS PILIMANUS (VON MARTENS)

Fig. 3. Parastacus pilimanus (Von Martens, 1869). Anatomical and histological characteristics of the genitalia of intersexes 1 and 2. A, B, anatomy of the
genitalia of the intersexes 1 and 2, respectively; C, D, histology of the gonads of intersexes 1 and 2, respectively; Sd, sperm duct; Od, oviduct; Te, testis;
Oo, oocytes; In, intestine; Ov, ovary; PCD, primary collecting duct; CT, collecting tubule; TA, testicular acini; Sz, spermatozoa; Oo, oocytes; FC, follicle
cells; Nu, nucleus; Yo, yolk. Scale bars = A, 3.0 mm; B, 2.0 mm; C, 120 μm; D, 130 μm.
81
82 ERICH H. RUDOLPH & ANA C. VERDI

TABLE I
Sex ratio by size range of Parastacus pilimanus (Von Martens, 1869), captured in the Province of
Maldonado, Uruguay

Size ranges Males Females Total Rate

(CL mm)
N % N %
10-14.9 2 26.8 5 71.4 7 0.4 : 1
15-19.9 12 46.2 14 53.8 26 0.9 : 1
20-24.9 14 48.3 15 51.7 29 0.9 : 1
25-29.9 5 45.5 6 54.5 11 0.8 : 1
30-34.9 4 36.4 7 63.6 11 0.6 : 1
35-39.9 3 30.0 7 70.0 10 0.4 : 1
40-44.9 3 42.9 4 57.1 7 0.8 : 1
45-49.9 0 0.0 1 100.0 1 —
Total 43 42.2 59 57.8 102 0.7 : 1

TABLE II
Results of comparisons between the cephalothorax length slopes and each one of the characters
measured in Parastacus pilimanus (Von Martens, 1869) intersex males and females, of a size greater
than 27.2 mm CL

Relation Slopes Student’s t-test

(t0.05(2),21 = 2.080)
Males intersex Females intersex
(N = 10) (N = 15)
CL × Abdominal somites L 0.897 0.869 3.429∗
CL × Somite 2 W 0.358 0.798 3.055∗
CL × Somite 3 W 0.320 0.908 3.022∗
CL × Somite 4 W 0.268 0.866 3.175∗
CL × Somite 5 W 0.208 0.930 2.875∗
CL × Somite 6 W 0.175 0.648 3.250∗
CL × Telson W 0.238 0.134 2.399∗
CL × Telson L 0.298 0.249 1.479
CL × Uropodal endopodite L 0.297 0.330 6.430∗
CL × Uropodal exopodite L 0.390 0.300 4.810∗
CL × Uropodal endopodite W 0.216 0.267 4.810∗
CL × Uropodal exopodite W 0.270 0.366 3.330∗
CL × Somite 3 pleuron H 0.095 0.091 7.240∗
CL × Somite 4 pleuron H 0.094 0.100 6.250∗
CL × Somite 5 pleuron H 0.098 0.110 6.320∗
CL × Somite 2 pleuron L 0.205 0.162 6.890∗

CL, cephalothorax length; L, length; W, width; H, height.

All t statistics indicated with ∗ , correspond to P  0.05.
 INTERSEXUALITY IN PARASTACUS PILIMANUS (VON MARTENS) 83

DISCUSSION

Female and male gonopores coexisted in all specimens of Parastacus pilimanus

analysed (N = 102), which was consistent with previous descriptions of supernu-
merary gonopores in this species by Von Martens (1869), Thompson (1982), and
Rudolph & Almeida (2000). Furthermore, sufficient records exist to qualify this
coexistence as constant in all species of Parastacus, with the exception of Paras-
tacus nicoleti. In this latter species, only a fraction of the populations present the
coexistence of male and female gonopores in the same individual (cf. Rudolph,
1995). As a consequence, with respect to this character, Parastacus pilimanus con-
forms to the diagnosis of Parastacus offered by Riek (1971) and Hobbs (1974,
1991). Nevertheless, many of the 102 specimens (N = 87; 85.3%) had sealed fe-
male gonopores with a semi-ellipsoidal aspect (intersex 1); while 14.7% (N = 15)
had ellipsoidal female gonopores, open to the exterior (intersex 2). This diversity
in form and sealing of the female gonopore also exists in other species of Paras-
tacus with supernumerary gonopores. Thus, in Parastacus pugnax, 57.8% of the
specimens analysed by Rudolph (1997) (N = 538) had female gonopores sealed
by a highly calcified cuticle. In a Uruguayan population of Parastacus varicosus,
93.7% of the individuals under study (N = 142) also had sealed female gonopores
(Rudolph et al., 2001). This diversity has also been described in Parastacus saffordi
Faxon, 1898, Parastacus brasiliensis, and Parastacus defossus, by Faxon (1898),
Almeida & Buckup (1997), and Noro et al. (2008), respectively.
Evidence gathered from anatomical dissections and histological sectioning
disclosed that, despite having coexisting male and female gonopores, specimens
of Parastacus pilimanus, have a male or female gonad from which gonoducts of
both sexes emerge, connecting it with the respective gonopores. In the ovaries,
it could be verified that their histology was uniquely female throughout, with no
traces of testicular tissue, and that oviducts were well developed, but sperm ducts
were rudimentary. On the contrary, the histology of the testes was entirely male
in the whole gland, with no trace of ovarian tissue, and sperm ducts were well
developed, while oviducts were rudimentary. This type of genitalia was already
observed in the first dissections of the reproductive system carried out in some
species of Parastacus (cf. Von Ihéring, 1892; Lönnberg, 1898; Turner, 1935), and
confirmed in more recent descriptions (Almeida & Buckup, 1997; Rudolph, 1997;
Rudolph et al., 2001). However, in the case of Parastacus pilimanus, this had only
been documented for the females of this species (Thompson, 1982).
The data provided by morphometric analyses of the abdomen and their com-
parison with evidence gathered from anatomical dissections, enables us to pos-
tulate that in individuals under 27.2 mm CL, there is only one type of external
morphology (intersex 1), making it very difficult to distinguish the sex of these
84 ERICH H. RUDOLPH & ANA C. VERDI

specimens externally. However, their gonads are already differentiated (54.8% in-
tersex females and 45.2% intersex males). The changes observed in the external
morphology of specimens over 27.2 mm CL, suggest that the gonadic females
around this size would undergo the puberty moult. This would enable the female
gonopores to complete their ellipsoidal form, develop setae on the external mar-
gin, and open to the outside (intersex 2), facilitating the extrusion of oocytes and
their posterior displacement towards the abdomen. Furthermore, the abdomen ac-
quires the appropriate morphology for egg incubation, i.e., greater relative width,
higher pleura, and a larger tail fan. According to our observations, as well as those
of Almeida & Buckup (2000) and Noro et al. (2008), these changes reflect the
maturing of the gonadic females. Each one of these morphological traits, taken
separately, and particularly together, permit the sex of specimens over 27.2 mm
CL to be distinguished externally.
Although the number of females is greater in all size classes where the sex ratio
was analysed, this difference could not be attributed to an eventual sex change,
given the presence of males in all of them, except between 45.9 and 49.9 mm CL.
This is not compatible with a protandric-type sex change unless this were partial,
which is not supported by our results, since no evidence of an ootestis was found
in any of the gonads analysed. The results of this study, together with previous
records of the permanent occurrence of gonopores and gonoducts of both sexes in
the same individual of Parastacus pilimanus (cf. Von Martens, 1869; Thompson,
1982; Rudolph & Almeida, 2000), suggest that this species presents permanent
intersexuality, similar to Parastacus pugnax and Parastacus varicosus in Uruguay.
Sex differentiation in the Malacostraca is controlled by the presence or absence
of the androgenic hormone. In the presence of this hormone, the gonadal primordia
are differentiated into male genitalia. In the absence of this hormone, ovarian auto-
differentiation occurs with female morphogenesis (Fingerman, 1987; Charniaux-
Cotton & Payen, 1988; Hasegawa et al., 1993; Bauer, 2000). In Parastacus
pilimanus, the permanent occurrence of oviducts and female gonopores in gonadal
males, and sperm ducts and phallic papillae in gonadal females, does not coincide
completely with the previously mentioned hormonal mechanisms. We have no
knowledge of the sex differentiation processes in Parastacus pilimanus and the size
or juvenile stage at which these occur. Neither have we identified the factor/factors
responsible for maintaining gonoducts and gonopores of one sex in individuals
whose gonad is of the opposite sex. We only know that these can be of different
types, because among other causes, the androgenic gland and its hormone are
controlled by a neuroendocrine gland: the X-organ-sinus-gland complex (Van Herp
& Payen, 1991). Similarly, we are unable to provide an answer with respect to the
adaptive value of a sexual system of this type. In order to provide such information,
and also to fill the previously mentioned gaps in our knowledge, we must await
 INTERSEXUALITY IN PARASTACUS PILIMANUS (VON MARTENS) 85

new studies that include larger samples from a greater number of populations,
in which, in addition to undertaking anatomical and histological analyses of the
sexual characters, the social organization and some physicochemical parameters of
the habitat are also studied, not only in this species, but in all species of Parastacus
that present permanent intersexuality.

ACKNOWLEDGEMENTS

The collaboration of the Research Department of the Universidad de Los Lagos,

for partially financing this study, and that of Susan Angus, for translating the
manuscript, are gratefully acknowledged.

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First received 15 September 2008.

Final version accepted 2 April 2009.