The structural basis for water exchange between the female cockroach (Blattella germanica) and her ootheca
Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0319, USA
* Author for correspondence: (e-mail: mullinsd{at}vt.edu)
Accepted 8 July 2002
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Summary |
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Key words: Blattella germanica, cockroach, water relations, oothecae, chorion
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Introduction |
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The mechanisms of oothecal formation, general structure
(Wheeler, 1889;
Ross, 1929
;
Wigglesworth and Beament,
1950
; Lawson, 1951
)
and embryonic development (Tanaka,
1976
) in cockroaches are available from earlier work. However,
Hinton (1981
) observed that
there is a need for more-detailed structural information, including
three-dimensional relationships of oothecal components. In this study, we have
examined some morphological and physiological aspects of the association of
B. germanica females and their oothecae in an attempt to provide new
information on the nature of this association.
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Materials and methods |
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Radiolabel studies
Whole-body transport
Two experiments were conducted to examine water-distribution patterns
between females and their oothecae. The females were either allowed access to
water (W) ad libitum or deprived of water (W/O) for 24 h. 500 nl
volumes of 3H2O (specific activity 1 mCi
ml-1, New England Nuclear, Boston, MA, USA) were injected into
abdomens of cold-immobilized (5°C) females carrying 11-20-day-old
oothecae. This was accomplished by using a 0.25 ml glass syringe with a 32
gauge needle and a motor-driven microapplicator. After incubation at time
intervals of 0.5 h, 2 h, 6 h or 24 h, the females were cold immobilized and
their oothecae detached. The oothecae were rinsed with distilled water,
blotted dry and placed on a glass microscopic slide mounted on a small block
of dry ice. After the oothecae were frozen, a chilled razor blade was used to
cut the oothecae into either equal quadrants (W) or sextants (W/O). The
females and respective oothecal sections were placed in separate
microcentrifuge tubes containing 500 µl distilled water and were then
subjected to a freeze(-70°C)/thaw(24°C) regimen three times before
homogenization with a microcentrifuge pestle. The homogenates were centrifuged
at 1000 g (5 min), and 250 µl of the supernatant was placed
in 8 ml Ecoscint scintillation fluid (National Diagnostics, Atlanta, GA, USA)
and analyzed using a Beckman LS-3150 scintillation counter (Beckman
Instruments, Inc., Irvine, CA, USA).
Permeability of the oothecal covering to water and water-soluble
molecules
The relative permeability of radiolabeled water-soluble molecules across
portions of the oothecal covering was determined using microparabiotic
chambers (Fig. 1). These
microparabiotic chambers were constructed from two pieces of glass tubing [2
cm length x 6 mm o.d./1.25 mm i.d.], each adapted with three tapered
catch hooks that were spaced equidistantly around their circumferences on one
end. In order to ensure a water-tight seal around the specimen, a 6 mm
parafilm gasket with a 1 mm pore punched into the center was placed on the end
opposite to the catch hooks of each chamber section. A 2 mm2
section of the oothecal covering, including the escutcheon-shaped vaginal
imprint area, was then removed from the proximal end of the ootheca and placed
between the two sections of the chamber. As the system was assembled, a larger
glass tubing sleeve [2 cm length x 9 mm o.d./6 mm i.d.] was placed over
the chamber ends, with the sample sandwiched between the parafilm gaskets.
Three dental rubber bands were then attached to the catch hooks to hold each
unit in place and ensure sufficient pressure to provide a good seal (see
Fig. 1). 30 µl of buffer
(10.3 g l-1 NaCl, 1.46 g l-1 KCl, 0.36 g l-1
NaHCO3, 0.21 g l-1 NaH2PO4
H2O, 1.34 g l-1 Na2HPO4 and 3 g
l-1 glucose; the pH was adjusted to 7.4 with 1 mol l-1
NaOH, as described by Kurtti and Brooks,
1976) was delivered to the chamber end facing the interior side of
the oothecal tissue, and 30 µl of the Kurtti and Brooks buffer containing
one of the radiolabeled materials listed below and fluorescein (50 µg
ml-1) was delivered to the chamber end facing the exterior side of
the oothecal tissue. At various time intervals (0 h, 1 h, 3 h, 6 h or 24 h), 1
µl buffer was removed from the interior side of the chamber for radioassay,
after which both exposed ends of the chamber were sealed with parafilm. The
chambers were examined microscopically to ensure that air pockets had not
formed between the tissue samples and the buffer solution. Samples removed
from each end of the chamber at various time intervals were delivered to 20 ml
scintillation vials containing Ecoscint scintillation fluid and were counted
using routine radioassay techniques. The following radiochemicals were used:
3H2O, specific activity 1 mCi ml-1=37 MBq
ml-1 (New England Nuclear); [U-14C]glucose, specific
activity 9.1 mCi mmol-1=337 MBq mmol-1 (ICN
Radiochemicals, Irvine, CA, USA), [1-U-14C]L-leucine, specific
activity 52 mCi mmol-1=1.92 GBq mmol-1 (ICN
Radiochemicals); [U-14C]formate, specific activity 56 mCi
mmol-1=2.07 GBq mmol-1 (ICN Radiochemicals);
[U-14C]glycine, specific activity 107 mCi mmol-1=3.96
GBq mmol-1 (Amersham Searle, Piscataway, NJ, USA) and
[14C]NaHCO3, specific activity 51 mCi
mmol-1=1.89 GBq mmol-1 (ICN Radiochemicals).
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Confocal microscopy
A 2-3 mm2 section of the proximal end, including the
escutcheon-shaped vaginal imprint area, was dissected from each oothecal
covering. Embryonic tissues were removed, leaving behind the chorion, which
tightly adheres to the internal surface of the covering. Samples were then
placed on a glass slide, surface stained with 1 µl fluorescein (5 mg
ml-1) and sealed in place with a coverslip. The preparations were
observed on a Zeiss LSM 510 Laser Scanning Microscope (software version 2.5)
using a Plan-Apochromat 100x objective, 488 nm argon laser and a BP
505-550 nm filter. A 5.6 µm stack was collected, and a three-dimensional
projection of the stack was generated.
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Results |
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Table 1 illustrates the distribution of 3H2O from hydrated females (W) through their oothecae over a 24 h period. Similar distribution of radiolabel through oothecae from water-deprived females (W/O) is provided in Table 2. Comparisons within groups (W and W/O) were based on the total amount of tritium detected in each female/oothecal system and are reported as the percentage of activity within each of the components. The rate of radiolabel remaining in the system after 24h was 75% (W) and 65% (W/O) of the injected dose, respectively (Tables 1, 2). Results indicate that over time, 3H2O moves from females through the proximal to the distal ends of their oothecae. Higher levels of activity were always found in the proximal end. Activity was distributed more or less evenly between the respective lateral quadrants/sextants of the oothecae. A comparison of the total amount of 3H2O contained in the oothecae from the W/O females (7.5%; Table 2) compared with that in the W females (18%, Table 1) 24 h after injection was significant [(P=0.01; analysis of variance (ANOVA)], indicating water availability to the female might influence the water-transfer process(es).
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SEM clearly revealed differences between the distal
(Fig. 2A) and the proximal
(Fig. 2B) ends of B.
germanica oothecae. The distal end has a relatively smooth surface, but
the proximal end contains an `escutcheon-shaped vaginal imprint' with
`delicate wrinkles' first described by Wheeler
(1889). Increasing the
magnification of the wrinkled region surrounding the vaginal imprint area
revealed that this region contains an area of small `pores' (approximately 1-2
µm in diameter) that penetrate the exterior of the oothecal covering
(Fig. 2C-G). The interior
surface of this region of the oothecal covering was also found to contain
pores that appear to penetrate the covering
(Fig. 2H,I). In order to
demonstrate that these pores penetrate completely through the oothecal
covering, we used a fluorescent stain that coated the external and internal
surfaces of the oothecal covering and a confocal microscopic system to examine
these two oothecal surfaces simultaneously. Confocal images generated by
optically scanning through a ventrolateral section adjacent to the escutcheon
confirmed that some of these pores do indeed penetrate the oothecal covering
(Fig. 3A-L). A
three-dimensional image showing proximal and distal aspects by rotation of a
reconstructed confocal stack also demonstrated that the pores are contiguous
(as represented by the black holes) with both the external and internal
surfaces (Fig. 3M-Q).
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To examine the permeability of water and water-soluble materials across the oothecal covering, a microparabiotic chamber was developed. Differences in water permeability between the distal and proximal ends of the oothecae were observed during preliminary experiments using this chamber. In these experiments, sections of the oothecal covering from distal and proximal ends of the oothecae were incubated for 24 h in the microparabiotic chamber assembly. A permeability of <5% of initial radioactivity was observed in distal samples compared with a permeability of 42% in proximal end samples. During development of the method, it became apparent that fluorescent dye was needed to ensure that leaks around the edges of the parafilm/tissue could be detected (Fig. 4). Samples showing evidence of leakage were excluded from data tabulations.
Results from experiments designed to compare the permeability of the oothecal escutcheon region to various radiolabeled water-soluble materials are presented in Fig. 5. These microparabiotic assays indicated that, although there were differences in permeability rate, the escutcheon region was permeable to all of the materials tested. In general, molecules with higher molecular weights (leucine, glucose, glycine and formate) showed significantly less movement across the escutcheon than did water or bicarbonate.
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Discussion |
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The striking physical differences between the proximal and distal ends of
B. germanica oothecae have been observed by numerous workers
(Lawson, 1951; Roth and Willis,
1954
,
1955a
;
Wheeler, 1889
). The distal end
of the ootheca is much darker and more highly sclerotized than the
more-flexible, unsclerotized proximal end, which can be almost white in color.
This obvious difference between the two ends led to studies on differential
permeability, water transfer and their relationship with relative humidity
(Roth and Willis,
1955a
,b
;
Barson and Renn, 1983
).
However, to our knowledge, no-one has investigated the structural differences
between the two ends of the oothecae in any detail, although Wheeler
(1889
) did report that the
proximal end of the oothecae typically contained an `escutcheon-shaped vaginal
imprint'. We have shown that the lateral ventral margins of the external
escutcheon contain pores of approximately 1-2 µm in diameter
(Fig. 2C-G). Furthermore, we
have confirmed that these pores penetrate the oothecal covering (Figs
2,
3).
The discovery of the pore field associated with the escutcheon supports
observations that the proximal end of the ootheca is more permeable to water.
Experiments using a microparabiotic chamber with radiolabeled materials
indicate that the escutcheon region is not only permeable to water but also to
low-molecular-weight water-soluble materials
(Fig. 5). This raises the
possibility that materials other than water might be transferred across the
proximal ends of the oothecae. In preliminary experiments, we found detectable
quantities of radioactivity in hatched nymphs from oothecae carried by gravid
females that had been injected with either radiolabeled glucose or leucine (D.
E. Mullins and K. R. Tignor, unpublished data). Additional work that might
indicate a mechanism for maternal investment in the oothecal development of
two other cockroach species (Byrostria fumigata and
Gromphadorhina portentosa) was done by Snart et al., who discovered
pores (approximately 1 µm in diameter) on papillae projecting from the
lumenal surface of female brood sacs
(Snart et al., 1984a).
Ultrastructural examination of these papillae revealed the presence of
`glandular units' associated with a duct that opens at a pore on the
cuticularised apex of the papillae (Snart
et al., 1984b
). This work raises some interesting questions on the
physiological, and perhaps nutritional, relationships that occur between the
female and the embryos that she carries in her brood sac during
gestation/embryogenesis.
There has been considerable work on the reproduction biology of the German
cockroach that includes both preovipositional and postovipositional aspects.
Preovipositional studies include vitellogenesis
(Martin et al., 1998),
hormonal regulation of oogenesis (Schal et
al., 1993
; Holbrook et al.,
2000
; Vilaplana, 1999), nutritional requirements associated with
reproduction (Cochran, 1983
;
Kunkel, 1966
) and maternal and
paternal investment (Mullins et al.,
1992
). Postovipositional (postovulation) investment and parental
care has been reviewed by Nalepa and Bell
(1997
). German cockroaches
appear to represent an important link in the evolutionary transition from
oviparity to ovoviviparity (Nalepa and
Bell, 1997
; Roth and Willis,
1954
). The process of oothecal formation is quite complex,
including chorion production, orientation and alignment of the eggs, their
encapsulation within the oothecae and elaboration of the keel
(Tanaka, 1976
;
Roth and Willis, 1954
;
Wheeler, 1889
). Formation and
protrusion of the oothecae is followed by a 90° rotation and retention of
the oothecae until after the time of hatching
(Tanaka, 1976
;
Roth and Willis, 1954
;
Wheeler, 1889
).
The attachment of the oothecae to the female during embryogenesis is
thought to be an initial step in development of ovoviviparity, a process
inclusive of oothecal formation followed by internalization within the female.
B. germanica may indeed represent an important intermediate stage
before internalization of the oothecae; there are at least four known species
of blattids that reproduce by ovoviviparity
(Roth, 1997). Ovoviviparity
(leading to viviparity) is thought to have appeared as an evolutionary
response for protection from biotic factors (mortality from predation,
pathogens, parasites, cannibalism), abiotic factors (avoidance of physical
extremes such as temperature, humidity, etc.) and selection of a suitable
habitat for the nymphs at the hatch. This is followed by development of
postovipositional support in terms of water exchange (ovoviviparity) and
provision of nutrients during embryogenesis (viviparity)
(Nalepa and Bell, 1997
).
The work presented here also raises some questions regarding the femaleembryo relationship(s). We have not yet examined the female vestibular structures that are in contact with the escutcheon while it is carried by the female. However, it is clear that the female genitalia clasp the oothecae quite firmly (Fig. 6), and the vestibulum that is associated with the proximal end of the ootheca appears to be membranous and capable of providing an environment in which water can be efficiently transported (Fig. 7). Fig. 2B shows that the porefield area associated with the perimeter of the escutcheon is about 0.5 mm2. The female genital area, which includes the vestibulum, appears to be large enough (1 mm x 2 mm; Fig. 6) to provide sufficient surface area for oothecal contact with the membranes lining the internal surfaces of the vestibulum (1 mm x 2 mm; Fig. 7), including the pore-field area of the ootheca. Close examination of the female vestibulum might provide useful information on the structural basis for liquid transport between the female and the oothecae that she carries.
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Another aspect regarding water transport relates to the mechanisms that are
involved within the ootheca itself. Transport is most likely achieved by the
chorion, but detail on how it is done is lacking. Also, there are differences
in interpretation of the structural architecture that have not been resolved
(Hinton, 1981). These
differences appear to reside in determinations of the composition and
arrangement of the components that comprise the hexagonal structure of the
chorion. Debate continues as to whether or not they are air filled
(Lawson, 1951
;
Wigglesworth and Beament,
1950
; Wheeler,
1889
; Hinton,
1981
). There is general agreement that the chorion contains
air-filled spaces that facilitate respiratory activities within the ootheca.
Access for respiratory exchange is through the spongy (white) bodies that are
apical extensions of the chorion and become isolated in the keel
(Hinton, 1981
). However, Lawson
(1951
) reported on an
`interesting sidelight' of his investigations resulting from the application
of dyed oil; he found that when dyed oil was applied near spongy (white)
bodies, it diffused from this area and gathered at the posterior ends of the
eggs. This observation supports the hypothesis that, in addition to gas
transport and exchange, the oothecal chorion, which envelopes each of the
embryos, might also provide for distribution of liquid.
Fig. 8 demonstrates that water
can be retained by both the hexagonal borders and by the internal structures.
Close examination of Fig. 8B
shows the pattern of water content as the water evaporates; the interior
portions of the hexagons clearing (dark areas) before those areas within the
hexagonal borders.
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Acknowledgments |
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References |
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