Hindawi Publishing Corporation
Psyche
Volume 2010, Article ID 428673, 10 pages
doi:10.1155/2010/428673

Research Article

Comparative Study of Spermatogenesis and Nucleolar Behavior in
Testicular Lobes of Euschistus heros (Heteroptera: Pentatomidae)

Hederson Vinicius de Souza and Mary Massumi Itoyama

Laboratório de Citogenética e Molecular de Insetos (LACIMI), Departamento de Biologia, Instituto de Biociências, Letras e Ciências
Exatas, Universidade Estadual Paulista (UNESP), Rua Cristóvão Colombo, 2265, Jardim Nazareth, CEP: 15054-000 São José do Rio
Preto, SP, Brazil

Correspondence should be addressed to Mary Massumi Itoyama, mary@ibilce.unesp.br

Received 23 March 2010; Accepted 17 June 2010

Academic Editor: Coby Schal

Copyright © 2010 H. V. de Souza and M. M. Itoyama. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

In some testicular lobes of the Pentatomidae there may be occurrence of atypical spermatogenesis or polymegaly, leading to
the production of nonfertile sperm. The comparative analysis of spermatogenesis and nucleolar behavior in testicular lobes of
Euschistus heros showed cells with polymegaly in lobes 4 and 6. Generally, when these lobes are present in the same individual,
there is also the formation of atypical cells in the flanking lobe. Such characteristic was not seen in E. heros. However, differences
regarding the concentration of heteropyknotic chromatin and silver-positive bodies in this lobe deserve attention. This study
explored the literature and demonstrated the prevalence of some lobes in the formation of differentiated cells. It was also found in
the literature that there is an association of the chromocenter with the nucleolus in several species of Pentatomidae, but in E. heros
this association does not appear to occur.

1. Introduction

The presence of testes formed by a number of compartments
referred to as “lobes” is a characteristic of the Heteroptera.
In some species, one of these lobes is of the harlequin type
that differs from the other lobes by showing spermatogonial
cells with meiotic pairing, nonspecific association of the
autosomal bivalents, anomalous arrangement of the chro-
mosomes in the metaphase plate, anomalous chromosome
segregation, and cell fusion, resulting in the production of
spermatozoa with highly variable chromosome numbers.
There are reports of this type of lobe in 15 genera in three
Pentatomidae subfamilies (Discocephalinae, Edessinae, and
Pentatominae) [1].

Other lobes may also be associated with the formation
of nonfertile sperm; for example, in Antiteuchus tripterus
(Pentatomidae), lobes 4 and 6 show cells with polymegaly
and significant intralobular metabolic differences [2, 3].

The aspects regarding the number of testicular lobes
and the formation of atypical sperm are little known and
explored. This information is found scattered in the literature

which makes it difficult to establish an evolutionary pattern
among testicular lobes with regard to the formation of fertile
and nonfertile sperm. For this reason, the data found in the
literature are compiled in Table 1.

Recent studies have demonstrated the importance of
examining the metabolic differences among species by the
analysis of nucleolar bodies. It is known that the nucleolus
or nucleolar bodies are related to the biosynthetic activity of
the cell, so that the size and number of bodies depend on the
functional characteristics of cells and may therefore reflect
metabolic and functional differences [3–7].

Another aspect of nucleolar behavior in spermatogenesis
is that, over time, several observations have suggested that
the nucleolar granules or bodies which persist at the end of
meiosis reorganize the nucleolus in early spermiogenesis and
support protein synthesis in the process [8].

The study of metabolic differences between testicular
lobes with analysis of nucleolar bodies has been proposed
by Souza et al. [3], who found that the testicular lobes
of A. tripterus showed significant differences in behavior
and size of the nucleolar bodies, which may be due to



2 Psyche

DR

PR

ED

(a)

1
2

3
4

5
6

(b)

Figure 1: Testis of Euschistus heros enclosed by red membrane,
where the proximal region (PR) of the ejaculatory duct (ED) is more
intense red than the Distal Region (DR) (a). Observe in (b) the
presence of six elongated lobes of approximately the same length,
with lobe 5 narrower than the others (arrow). Bar = 1 mm.

differences in the formation of sperm with a nonfertile
function.

The analysis of nucleolar organizer regions (NORs)
during prophase has shown a close association of the
chromocenter with the nucleolar body [9, 10]. The chromo-
center in Heteroptera is characterized by its heteropyknotic
nature, where it can be composed of sex chromosomes or
even by heterochromatic autosomes [9, 11, 12]. In general,
the nucleolar body is disorganized during diakinesis [13],
reorganizing itself only in the beginning of spermiogenesis,
supporting the initiation of protein synthesis [8]. How-
ever, the relationship between chromatin heteropyknotic
and nucleolar bodies during spermiogenesis has not been
previously explored, according to the literature.

Thus, the objective of this study was to analyze sper-
matogenesis in each lobe of Euschistus heros, comparing it
with nucleolar behavior throughout spermatogenesis and
to analyze in detail the distribution pattern of testicular
lobes, described in the literature, with regard to differentiated
spermatogenesis.

2. Material and Methods

Fifteen adult males of Euschistus heros Fabricius, 1794 (Het-
eroptera, Pentatomidae, Pentatominae, Pentatomini) were
collected on soybean plants (Glycine max (L.), in the city of
Sao Jose do Rio Preto (20◦47′13′′ S, 49◦21′38′′ W), SP, Brazil.
The insects were fixed in methanol:acetic acid (3 : 1), and
their testicular lobes were separated and submitted to the
squash technique with lacto-acetic orcein staining, which is
chromosome specific.

To study nucleolar behavior during spermatogenesis, the
slides were submitted to the silver impregnation technique
[47, with modifications] to stain argyrophilic proteins, which

are associated with rRNA and which can therefore localize
the nucleolus or nucleolar bodies.

In the statistical analysis, measurements were taken of
the diameter of 100 cells and their nuclei in the diffuse
stage, that is, the stage after pachytene, which is characterized
by its increased size and chromatin scattered throughout
the nuclei, and of 100 cells nuclei in the stage of round
spermatid in each lobe, randomly chosen. We used the
program UTHSCSA Image Tool v.3.00 [48] and Minitab
version 15.1 [49] for ANOVA (Tukey’s comparison with
95% confidence interval) to compare the measurements of
the cells between the testicular lobes. The best images were
captured with a Zeiss microscope using the image analysis
program AXIO VISION.

3. Results

3.1. Testis Morphology. The testes of Euschistus heros were
enclosed by peritoneal sheath with red pigment, with the
proximal region (PR) of the ejaculatory duct (ED) being
more pigmented than the distal (DR) (Figure 1(a)). When
the peritoneal sheath was removed in the distal region,
we could observe the presence of 6 elongated lobes of
approximately the same length, where lobe 5 was narrower
than the others (Figure 1(b)).

3.2. Meiotic Behavior. The comparative analysis of meiotic
cells of Euschistus heros stained with lacto-acetic orcein and
silver impregnated showed that the behavior of cells in the six
testicular lobes was quite similar, and therefore, the results
are presented together.

During early prophase, a heteropyknotic body was
observed at the periphery of the nucleus, and variation in
cell diameter among the lobes was the only difference found.
The diameter of the cells in lobes 1–3 (Figure 2(a)) was
significantly smaller than that of lobe 5 cells (Figure 2(c)),
which were smaller than cells in lobes 4 and 6 (Figure 2(b)).
Due to these differences, an ANOVA test was performed,
which showed that the lobes could be grouped by the size
of the cells and their nuclei (Figures 2(a)–2(c)) into three
different groups (group 1 = lobes 1, 2, and 3, group 2= lobe
5, group 3= lobe 4 and 6). Significant differences (P < .0001)
were found between the groups, and the cells of group 1
(lobes 1–3) and their nuclei were smaller than those in group
2 (lobe 5) which, in turn, were smaller than those of group 3
(lobes 4 and 6) (Table 2).

During spermatogenesis, it was observed that the cells in
diplotene/diakinesis showed chromosomes with chiasmata
(Figure 2(d)). It was possible to determine in diakinesis
and metaphase I the presence of a diploid number of
2n = 14 (12A + XY) chromosomes (Figures 2(d), 2(e)). In
anaphase/telophase I, it was observed that only autosomes
undergo reductional segregation (Figures 2(f), 2(g)). During
metaphase II, the autosomes were arranged in a ring with
the sex chromosomes arranged inside (Figure 2(h)). Dur-
ing anaphase/telophase II, the sex chromosomes undergo
equational division and it was possible to visualize lagging
migration of the X chromosome (Figure 2(i)).



Psyche 3

Table 1: All species of the family Pentatomidae in the literature where authors noted the number of testicular lobes and if they exhibited
atypical meiosis and polymegaly. “No”: characteristic not found; “—”: information not found, “?”: author not sure about the presence of the
characteristics.

Classification No. lobes Atypical meiosis Polymegaly References

Pentatomidae family

Subfamily Asopinae

Apateticus crocatus 7 No No [14]

Euthyrhynchus floridanus (Linnaeus) 6 No No [14]

Perillus bioculatus (Fabricius 1775) (as Mineus
bioculatus, 1775)

7 No No [14, 15]

Podisus maculiventris (Say) 7 No No [14]

(as P. modestus (Dallas, 1851)) [15, 16]

(as P. spinosus (Dallas, 1851)) [15–19]

Stiretrus anchorago (Fabricius 1775) 7 No No [14, 16]

Subfamily Discocephalinae

Tribe Discocephalini

Antiteuchus tripterus (Fabricius,1787) (as
Mecistorhinus tripterus)

6 5 4 and 6 [2, 20–22]

Dinocoris rufitarsus (Ruckes, 1958) 8 5 4 and 6 [21, 23]

Discocephalessa humilis (Herrich-Schaeffer,
1843) (as Platycarenus notulatus (Stal, 1862))

4 No — [20]

Platycarenus umbractulatus 7 No — In press

Tribe Ochlerini

Alitocoris schraderi (Sailer, 1950) 5 5 4 degenerate [21, 24]

Subfamily Edessinae

Brachystethus rubromaculatus (Dallas, 1851) 4 4 — [20]

Edessa bı́fida (Say) 5 No 2 and 4 [14]

E. meditabunda (Fabricius, 1794) 4 No — In press, [25]

Subfamily Pentatominae

Tribe Aeliini

Aelia americana 7 No No [14]

Tribe Carpocorini

Carpocoris sp. 6 No 3 ? [14]

Coenus delius (Say, 1832) 6 5 4 and 6 [14, 15, 17–19]

Cosmopepla bimaculata (Distant) 5 No ? [14]

Euschistus euschistoides, (Vollenhoven, 1868)
(as E. ssilis Uhler, 1871)

6 5 4 and 6 [14, 15, 19]

Euschistus heros (Fabricius, 1798) 6 No 4 and 6 Present work

E. ictericus (Linnaeus, 1763) 6 5 4 and 6 [14, 15]

E. inflatus 6 5 4 and 6 [14]

E. servus (Say, 1832) 6 5 4 and 6 [14, 15, 25, 26]

E. tristigmus (Say, 1832) 6 5 ? 4 and 6 [14, 15, 17, 18, 26]

E. variolarius (Palisot de Beauvois, 1805) (as
Pentatoma)

6 5 4 and 6
[14, 15, 17, 18, 27–

30]

Holcostethus limbolarius (Stål, 1872) (as
Peribalus)

6 No No [14, 17, 18]

Mormidea quinqueluteum (Lichtenstien, 1796) 3 No — [1, 9]

Oebalus poecilus 4 No — [9]

O. (Fabricius, 1775) (as Solubea pugnax) 4 No No [14, 16, 26]

O. ypsilongriseus (De Geer, 1773) 4 No — [9]



4 Psyche

Table 1: Continued.

Classification No. lobes Atypical meiosis Polymegaly References

Trichopepla semivittata (Say) 7 No No [14, 17, 18]

Tribe Chlorocorini

Arvelius albopunctatus (De Geer, 1773) 6 4 3 and 5 [1, 14, 31]

Chlorocoris complanatus 7 5 4 and 6 In press

Loxa flavicollis (Drury, 1773) (as L. florida (Van
Duzze))

7 5 4 and 6 [30, 32, 33]

L. viridis (Palisot de Beauvois, 1805) (as L.
picticornis (Horvath, 1925))

7 5 4 and 6 [21, 32, 33]

Tribe Nezarini

Chlorochroa uhleri (Stål) 6 No 3 and 5 [14]

Nezara viridula (Linnaeus, 1758) 6 4 3 and 5 [14, 34–39]

Rhytidolomia saucia (as Chlorochroa saucia)
(Say, 1832)

6 No 3 and 5 [14, 40]

R. senilis (as Chlorochroa senilis) (Say, 1832) 6 3 and 5 No [14, 40, 41]

Tribe Halyini

Brochymena quadripustulata (Fabricius) 7 No 4 and 6 [14]

Tribe Pentatomini

Acledra hilare (Say, 1832) (as Nezara hilaris) 6 No No
[14, 15, 17–
19, 34, 38]

Adevoplitus longicomis (Ruckes, 1958) (as
Pseudevoplitus longicomis)

6 — 3 and 5 [21, 41]

Banasa calva (Say, 1832) 3 No No [14, 42–44]

B. dimidiata (Say, 1832) 3 No No [14, 43, 44]

Thyanta calceata (Say, 1832) [as T. custator
(Fabricius, 1803)]

4 3 ? No [14, 31, 34]

T. casta 6 No 3 and 6 [14]

T. custator 4 3 ? No [14]

T. perditor (Fabricius, 1794) 3 No — [45]

Tribe Strachiini

Murgantia histriônica 5 No 3 and 4 [14]

M. histriônica (var. nigricans) 5 No 3 and 4 [14]

Tribe Piezodorini

Piezodorus guildinii (Westwood) 5 ? No No [14, 46]

Table 2: Mean diameter of cells in diffuse stage and their respective
nuclei and round spermatids of Euschistus heros, chosen randomly.
The lateral bars indicate different groups with equality average. The
values are given by Mean ± standard deviation (Mean ± SD). The
unit utilized is micrometers (µm).

Values (µm)

Lobes Cell Nuclei Spermatids

1 23.60± 0.20 16.68± 0.15 8.32± 0.06

2 23.51± 0.22 17.45± 0.15 8.72± 0.06

3 23.06± 0.24 15.79± 0.20 8.53± 0.05

4 26.08± 0.25 19.53± 0.17 8.64± 0.09

5 37.78± 0.43 27.70± 0.28 14.15± 0.10

6 42.72± 0.43 30.28± 0.29 13.27± 0.11

P < .0001 P < .0001 P < .0001

When cells in prophase were silver impregnated, three
round silver-positive bodies could be seen, two more
impregnated with one bigger than the other, and the
third lighter staining (Figures 2(j), 2(k)). It could also
be observed that the lighter body may correspond to the
heteropyknotic body revealed by the lacto-acetic orcein
technique (Figures 2(a)–2(c)). In addition to the increased
size of the cells in prophase of lobes 4 and 6, there were
also several silver-positive bodies scattered throughout the
nucleus (Figure 2(k)). With regard to metaphase I, two
different behaviors were observed: lobes 1, 2, 3, 4, and
6 with impregnations in the cytoplasm and chromosomes
(Figure 2(m)) and lobe 5 which showed negative silver
impregnation (Figure 2(n)). During anaphase/telophase II,
the positive silver impregnation behavior was similar for



Psyche 5

(a) (b) (c)

(d) (f) (g)

(h)

(e) (i)

(o)(n) (p)(m)

(l)(j) (k)

Figure 2: Meiotic cells stained with lacto-acetic orcein ((a)–(i)) and silver impregnated ((j)–(p)). ((a)–(c)) Early prophase I with
heteropyknotic body in the periphery of the nucleus (arrows), with (a) belonging to lobes 1–3, (b) to 4 and 6 and (c) to 5; (d)
diplotene/diakinesis showing a bivalent with interstitial chiasma (arrowhead) and heteropyknotic body (arrow); (e) final stage of diakinesis
showing X and Y chromosome, respectively, arrow and arrowhead; ((f), (g)) anaphase/telophase I with regular segregation of chromosomes;
(h) metaphase II showing the autosomes arranged in ring-shape and the sex chromosomes in the center; (i) anaphase/telophase II with
lagging migration of the X chromosome (arrow) and the Y chromosome in the opposite part of the cell (arrowhead); ((j)–(l)) early prophases
I showing two round silver-positive bodies (arrows) that are disorganized during the subsequent phases and one body less impregnated
(arrowheads). Note that in prophase of lobes 4 and 6 (k), there are several silver-positive bodies scattered throughout the nucleus; (m), (n))
metaphase I with silver impregnation in the cytoplasm and chromosomes (m), differing from lobe 5, which has silver-negative metaphase
(n); ((o), (p)) telophase II with nucleolar reorganization in both cell formations (arrows). Bar = 10 µm.

all lobes, that is, nucleolar reorganization in both cells in
formation (Figures 2(o), 2(p)).

3.3. Behavior of Cells during Spermiogenesis. A comparative
analysis of the cells during the spermiogenesis of E. heros

stained with lacto-acetic orcein and silver impregnated
showed that the behavior of cells in six testicular lobes was
similar, and therefore, the results were presented together.

In the round spermatid, heteropyknotic staining was
observed at the periphery and center of the nucleus, and



6 Psyche

(a) (b) (c) (d)

(f) (g) (h)(e)

(i)

(o)(n)

(s)(r) (t)

(q)

(p)

(m)(l)(j) (k)

Figure 3: Spermiogenesis cells stained with lacto-acetic orcein ((a)–(h)) and silver impregnated ((i)–(t)). (a)–(d) Development of spermatids
in lobes 1–3 and 5. (a) Round spermatids showing heteropyknotic material at the periphery and center of the nucleus (arrows) and a vesicle
in the posterior region (arrowhead); (b) spermatid in elongation with heteropyknotic material at the periphery of the nucleus (arrow); (c)
in a following stage, the heteropyknotic chromatin is visualized on only one side (arrow); (d) in a later stage, it becomes indistinguishable;
(e)–(h) spermatids of lobes 4 and 6; (e) round spermatids showing a vesicle in the posterior region (arrowhead); (f) spermatid in elongation;
(g) spermatid at the later stage of development with chromatin stained weakly and in the posterior region with a protuberance (arrow); (h)
sperm showing a thin heteropyknotic chromatin on one side of the head (arrow); ((i)–(m)) round spermatid with several silver-positive
bodies; ((j)–(l)) during the subsequent phases were observed three silver-positive bodies: one rod-shape and lighter close to region of tail
formation ((j), smaller arrow), one round intensely stained (arrowhead), both in the posterior region of the spermatid and another smaller
than others (bigger arrow) that was visualized in the middle region of the spermatid ((k), (l)); (m) spermatid in development with only one
silver-positive body in the posterior region of the nucleus (arrow); (n) spermatozoon of lobes 1–3 with continuous silver-positive staining in
the posterior region of the head (arrow) and in lobe 5 (o) a continuous positive silver impregnation (arrow) and several small bodies in all
the nucleus (arrowhead). ((p)–(t)) Spermiogenesis of lobes 4 and 6; (p) spermatid in development showing strong silver-positive body in the
posterior region of the head (arrow), several small silver-positive bodies in the middle region and a line-shaped silver staining from middle
to anterior region (arrowhead); ((q)–(t)) developed spermatids showing four silver-positive behaviors: (q) continuous and linear (arrow);
(r) large and amorphous; (s) linear and throughout the extent of the head and several round and small silver-positive bodies and (t) with the
same previous characteristics, but the silver-positive bodies are larger and smaller in number. Bar= 10 µm.



Psyche 7

a vesicle was seen in the posterior region (Figure 3(a)); in
the elongated spermatid, the heteropyknotic material could
be visualized at the periphery of the nucleus (Figure 3(b)).
In a following stage of the development of the spermatid,
the heteropyknotic chromatin could be observed on only
one side (Figure 3(c)) while in a later stage becomes
indistinguishable (Figure 3(d)). Despite the behavior of the
spermatids in early development being similar among all six
lobes, it was observed, in regard to the diameter of round
spermatids, that the lobes could be grouped into two, by the
size of cells (group 1= 1–3 and lobe 5, group 2= lobes 4, and
6). The cells of group 1 (lobes 1-3 and 5, Figure 3(a)) were
significantly smaller than those in group 2 (lobes 4 and 6,
Figure 3(e)) (P < .0001) (results summarized in Table 2).
The same development pattern of cells in spermiogenesis
was observed of lobes 4 and 6 in relation to the other lobes;
however, the heteropyknotic chromatin in early spermatids
was less evident (Figures 3(e), 3(f)). In the developed
spermatid, it is inconspicuous, where a protuberance appears
in the posterior region of the nucleus (Figure 3(g)) and
fine chromatin along the head of the sperm in formation
(Figure 3(h)).

With the use of silver impregnation in the cells of
spermiogenesis, round spermatids were observed with sev-
eral round silver-positive bodies (Figure 3(i)), which moved
to the posterior region of the nucleus, near the tail in
formation (Figure 3(i)) and during the development of
the spermatid (Figure 3(j)–3(l)), three silver-positive bodies
were observed: one rod-shape and lighter located at the
beginning of the formation of the tail, and two round ones
of different sizes and intensities of impregnation. The largest
and least impregnated was located in the posterior region
of the spermatid’s nucleus, close to the rod-shaped body,
and the smaller and most impregnated was close to the
anterior region of the spermatid’s head (Figure 3(j)). With
the development of the spermatid, apparently, these bodies
remain in the same locations (Figure 3(j)–3(l)). During later
stage of development there was only one silver-positive body
in the posterior region of the nucleus (Figure 3(m)).

Differences were seen among the lobes in the pattern of
silver impregnation in the developed spermatid. In lobes 1–3,
this spermatid showed only a continuous silver impregnation
in the posterior region of the head (Figure 3(n)), while in
lobe 5, besides showing this continuous impregnation in
the same region, also contained several small silver-positive
bodies in the entire nucleus (Figure 3(o)). The developed
spermatids in lobes 4 and 6 exhibited intense and elongated
silver impregnation in the posterior region of the head,
several small silver-positive dots in the middle region, and
a line of silver staining from the middle region up to the
anterior region of the head (Figure 3(p)). In the elongated
spermatid, four different behaviors were noted in regard to
distribution of silver impregnation: continuous and linear
(Figure 3(q)); large and amorphous (Figure 3(r)); one linear
region in the entire extent of head and several small dots,
distributed throughout the head (Figure 3(s)); one linear
region in the entire extent of head, several small dots, and
larger round stained bodies distributed throughout the head
(Figure 3(t)).

4. Discussion

The testes of Pentatomidae are divided by connective tissue
into subunits called “lobes”. The most common number of
lobes is seven, although there are variations among tribes and
species [1, 50]. Based on the literature review regarding the
number of lobes (Table 1), it could be seen that among the six
species of the subfamily Asopinae, only one (Euthyrhynchus
floridanus) has six testicular lobes, while the others have
seven. In the other subfamilies, the distribution of lobes was
found to be heterogeneous, ranging from four to eight lobes
in the Discocephalinae, four to five in the Edessinae, and
three to seven in the Pentatominae. At this moment, it is
not possible to establish a direct relationship between the
number of lobes and subfamily. Unfortunately, despite that
the Heteroptera are composed of many species, very few
have been studied in regard to this aspect. The subfamily
Pentatominae, the most studied, showed wide variation with
relation to the number of lobes. Therefore, a larger number
of species should be analyzed to determine the ancestral
number of lobes as well as to understand this diversity in the
number of lobes.

Another feature found in species of the family Pentato-
midae is the presence of a different lobe called harlequin.
Schrader [21, 23, 32] was surprised that the forces of evolu-
tion have persisted with the harlequin lobes, a structure that
produces heteropolyploid sperm not used in fertilization.
They suggested that this sperm should provide additional
nutrients, especially nucleoproteins for the development of
eggs.

Atypical meiosis is another unusual feature that can be
observed in the harlequin lobes. Table 1 shows the prevalence
of these lobes in the subfamilies Discocephalinae, Edessinae,
and Pentatominae. Among the six species of the subfamily
Asopinae, no distinguishing characteristic was mentioned.
However, there are no data in the literature with regard
to the number of lobes and formation of atypical cells
for the other subfamilies (Cyrtocorinae, Phyllocephalinae,
Podopinae, and Serbaninae).

It could also be observed from the analysis of 50 species
in Table 1 that 19 (38.0%) have atypical meiosis, of which 14
(73.7%) correspond to lobe 5, three (15.8%) to lobe 4, and
three (15.8%) to lobe 3. It was found only one occurrence
involving the lobes 3 and 5 in the same individual.

In addition to this atypical meiosis, the production of
different-size sperm from the normal meiotic process also
presents an evolutionary mystery. Although many species
of insects have sperm showing polymegaly, which vary only
in size and have a normal chromosome complement, all
forms of sperm morphology may not be equally involved
in fertilization [51]. The capacity of fertilization in sperm
of different morphologies has not been demonstrated. At
least for the large sperm, a nutrition argument similar to
proposed to harlequins sperm [21] could be advantageous to
the formation of multiple sperm morphology.

In Table 1, polymegaly was described in 25 (50.0%)
species, of which 14 (56.0%) correspond to the lobes 4
and 6 simultaneously, that is, both lobes showed cells with
polymegaly in the same individual, and only one (4.0%)



8 Psyche

case not involving simultaneity in the formation of cells with
polymegaly was found for lobes 3 and 4. These simultaneous
events involving lobes 2 and 4, 3 and 4, 3 and 5 and 3 and
6 occurred in 1 (4.0%), 2 (8.0%), 5 (20.0%), and 1 (4.0%)
cases, respectively.

When analyzing these two characteristics, the formation
of atypical cells and polymegaly, it could be seen in
Table 1, that when there are two lobes in the same species
showing polymegaly, these lobes usually flank another lobe,
as occurs in the species Antiteuchus tripterus. Especially
for lobes 4 and 6, in which 12 simultaneous events
were observed, only one did not show lobe 5 (flanking
lobe) with the formation of atypical cells. This exception
corresponds to species Euschistus heros, analyzed in this
study. Although there was no formation of atypical cells in
lobe 5, differences were found in the diameter of cells in
prophase and in the concentration of silver impregnation,
mainly in the sperm, suggesting the necessity to explore
the true function of the sperm in this lobe, using other
approaches.

Table 1 indicates that the minimum number of lobes
found for the family Pentatomidae is three, as occurs in
the species Mormidae quinqueluteum [1] and Banasa calva
[43]. When a species possessed lobes forming differentiated
cells (atypical or showing polymegaly), there was also the
presence of at least three lobes demonstrating the production
of fertile sperm, as in species A. tripterus [2] which has six
lobes, three lobes responsible for producing typical sperm
and the other three producing differentiated cells. Therefore,
it could be inferred from the data obtained to date, that
three is the minimum number of lobes needed to produce
fertile sperm. Another feature that could be shown among
these analyzed species described in Table 1, was that lobe
1 invariably produces fertile sperm, that is, with meiosis
and production of typical sperm, while the only case of
production of differentiated cells involving lobe 2 occurred
in the species Edessa bifida [14].

When formation of differentiated cells occurred, this was
related to lobes 4 to 6. This may suggest a preadaptation to
the formation of nonfertile sperm in these lobes. However,
with the data obtained so far, it has not been possible to
characterize a distribution pattern of evolution of nonfertile
sperm between the subfamilies or tribes.

The species E. heros showed cells in the diffuse stage
in lobes 4 and 6, significantly larger than in other lobes,
and therefore considered polymegaly. Morphologically, lobe
5 was narrow, in contrast to that in the species A. tripterus
studied by Souza et al. [2]; lobes 4 and 6 produced cells with
polymegaly, but lobe 4 was the narrowest in this species,
while the lobe 5 (harlequin lobe) was disproportionately
larger, forming a slightly twisted testis.

The differences found in regard to heteropyknotic
material and silver impregnation in the lobes of E. heros
showed that polymegaly is accompanied by differences in
the behavior of these structures, especially when compar-
ing spermiogenesis. Intensive silver impregnation and no
condensed chromatin in spermatids and sperm may reflect
intense metabolic activity and suggest a change in function
in the sperm of these lobes.

Regarding cytogenetics, in Heteroptera, there is generally
a close association of the chromocenter with the nucleolus in
prophase I. Souza et al. [9] found an unusual morphology
of the nucleolar body, called mushroom, which supports
the association of the chromocenter, probably the more
stained portion (the cap) with the nucleolus (the stem). The
species of this study showed a heteropyknotic body when
stained with lacto-acetic orcein, and when silver impreg-
nated showed two darker bodies which were probably the
nucleolar bodies, and a lighter one which was probably the
chromocenter. It is interesting to note that the chromocenter
may not be associated directly with the nucleolus in E. heros,
diverging from other species of the Pentatomidae that show
this association [9].

Thus, this paper provides insights into evolutionary
process of the formation of nonfertile sperm, once this field
lacks a more accurate exploration of such theme by different
approaches.

Acknowledgments

Special thanks go to Dr. Sonia Maria Oliani of the Depart-
ment of Biology of IBILCE/UNESP for the opportunity to
capture cell images. Dr. Jocélia Grazia of the Department
of Zoology of Universidade Federal do Rio Grande do
Sul and Dr. Aline Barcellos of the Museu de Ciências
Naturais of Porto Alegre, Brazil who helped with specimen
identification. Research supported by Foundation for the
Development of Sao Paulo State University (FUNDUNESP),
Foundation for Research Support of the State of São
Paulo (FAPESP) and National Counsel of Technological and
Scientific Development CNPq.

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