Regional Differences in Methyl Farnesoate Production by the Lobster Mandibular Organ

8 Pages·1994·3.2 MB·English

by  D W Borst

Reference: Biitl. Bull 186: 9-16. (February.
Regional Differences in Methyl Farnesoate Production
by the Lobster Mandibular Organ
DAVID W. BORST1, BRIAN TSUKIMURA1, HANS LAUFER2, AND ERNEST F. COUCH3
Department ofBiology. Illinois State University, Normal. Illinois 61790-4120: Department of
Cellular Biology, University ofConnecticut, Slorrs, Connecticut 06268: and ^Department ofBiology,
Texas Christian University, Fort Worth. Texas 76129
Abstract. Visual examination ofthe mandibular organ a sesquiterpene structurally related to the insectjuvenile
(MO) from the lobster, Homarus americanus. disclosed hormones (JH) (Borst et a/., 1987; Laufer et at.. 1987;
two distinct morphological regions: a fan-folded region Tobe et at.. 1989; Borst and Laufer. 1990). Thissynthetic
MO
alongoneedgeofthegland,andasmooth, unfolded region activity is consistent with ultrastructure, which has
comprising the rest ofthe gland. Because MOs produce featurestypical ofendocrinecellsproducing lipidand ste-
MF
methyl farnesoate (MF). the content ofboth regions roid hormones (Aoto et a!.. 1974; Byard et a/.. 1975; De-
wasmeasured. In freshlydissectedglands, morethan 95% meusy, 1975; Hinsch and al Hajj, 1975; Hinsch, 1977;
MF
ofthe wasfound in thefan-folded region ofthegland. Buchholz and Adelung. 1980). Similar ultrastructural
MO
In sections incubated with 3H->mv/n'/]methionine features are observed in the insect corpus allatum (CA)
(a radiolabeled precursor ofMF),[ more than 90% ofMF (Cassier, 1979; Tobe and Stay, 1985), the site ofJH syn-
synthesis was found in the fan-folded region. Eyestalk thesis. These similarities of ultrastructure and of bio-
MO MO
ablation, a procedure that increases activity, caused chemical product suggest that the crustacean is the
the MF content ofMOs to increase more than 130-fold, homologue ofthe insect CA.
but had little effect on the regional distribution of MF. Although MF is a major MO product, several reports
Histological observationsindicated that thesetwo regions have suggested that this tissue produces other endocrine
had different cellular compositions. The fan-folded region products. For example, significant amounts ofprogester-
contained two cell types (A and B). The A cells were mi- one have been detected in lobster MOs by radioimmu-
totically active and appeared to be undifferentiated. The noassay(Couch etat.. 1987). Moreover, lobsterMOscon-
B cells contained a large number ofsmall vacuoles. The vert pregnenolone to progesterone in vitro (Tsukimura,
unfolded region was largely composed ofa third cell type 1988). MOs from other species, such as the mud crab
(C). TheCcellswere largeand morphologically complex, Scylla serrala and the crayfish Procambarus clarkii, pro-
containing many mitochondria and large vacuolar-like duceand release farnesoicacid in vitro(Tobe et al.. 1989;
structures. They contained relatively few small vacuoles. DingandTobe, 1991). However, theimportanceofeither
On the basis ofappearance and location, B cells appear compoundasasecretory product //; vivoisstill uncertain.
to be the likely site of MF synthesis. The physiological Because of its large size, the lobster MO is an ideal
importance ofC cells is unknown. tissue for investigating MO function (Byard et a/.. 1975).
In this paper we describe studies on the structure and
Introduction function ofthis tissue.
Mandibular organs (MOs) from several crustacean Materials and Methods
decapodshavebeen shown toproduce methyl farnesoate.
Received 20July 1992:accepted 22 November 1993. Male lobsters(200-300 g)were obtained from the Ma-
Abbreviations: MO = mandibular organ; MF = methyl farnesoate: rine Resources Department at the Marine Biological Lab-
EF= ethyl farnesoate. oratory, Woods Hole, Massachusetts. Animals were kept
10 D. W. BORST ET AL
in runningseawateratambient temperature (15 to 20C) gentleshakingin Homarussaline(WelshandSmith, 1960)
until used. The eyestalks of some animals were severed supplemented with glucose (0.1%) and 200 pM pH-
MF
at their base; bleeding was limited by cauterization. An- /mv/;r/]methionine. After two hours, was extracted
imals were eyestalk-ablated 2 weeks before use. from the tissue and the incubation medium.
MF
These extracts were then used to determine syn-
Materials thesis by two different methods. In one approach. np-
Acetonitrile and diethyl ether(both HPLC grade), and HPLC was used to calculate the amount of MF synthe-
hexane (Optima grade) were purchased from Fisher Sci- sized during the incubation of MOs. This method is fa-
MO
entific (Pittsburgh, PA). [3H-;i'//n7]methionine (specific cilitated bytheobservation that freshlydissected paMirFs
activity = 200 mCi/mmol) was obtained from Dupont/ from the same animal contain similar amounts of
NEN (Boston, Massachusetts). JB-4 glycol methacrylate (see Results, Fig. 2). The MFcontained in incubatedgland
was purchased from Polysciences, Inc. (Warrington, sections and their culture media was quantified by
Pennsylvania). MFwasobtained from Dr. D. A. Schooley npHPLC. The sum ofthese valueswasthen compared to
MF
(Zoecon Research Institute, Palo Alto, California) as a the found in sectionsofthe unincubated contralateral
mixture oftwo isomers (approximately 70% 2E,6E and pair. The difference between these values is the amount
MF MO
30% 2Z,6E). The 2E,6E isomer was purified by normal of synthesized by the during the incubation.
MF
phase high performance liquid chromatography (np- In a second method, the relative level of synthesis
HPLC; see below). Ethyl farnesoate was prepared from wasdetermined by calculating the transferofradiolabeled
MF
2E,6E methyl farnesoate as previously described (Borst methyl groupsfrom [3H-/Ht7/n'/]nietnionineto during
eial, 1987). incubation (Borst el al. 1987; Lauferf/a/., 1987). Because
lobster MOs synthesize and release more than one radio-
Mandibular organ dissection labeled organosoluble compound (Borst el al., 1987), the
MO
MOswere removed from the area adjacent to the apo- hexane extracts of sections and their culture media
deme ofthe mandibular abductor. Some MOs were cut were fractionated usingthe npHPLC conditions described
transversely witha razor blade into fourstrips orsections above. The radioactive material eluting at the retention
MF
(I-IV) ofapproximately equal width (see Fig. 1). In a few time of was quantified with a liMquFid scintillation
cases, each section was furtherdivided into three or more counter. The sum of the radiolabeled in the tissue
pieces of equal size by cutting perpendicular to the sections plus that released into their culture medium is a
first cut. measure ofMF synthesis by the MO. While this in vitro
MF labeling procedure is more sensitive than the npHPLC
quantification procedure used above, it can be affected by factors other
Whole MOs or MO sectionswere homogenized in dis- than the absolute synthetic rate of the MO. Therefore,
MF
tilledwaterwith aDounce homogenizer, the homogenizer the results are relative indicators ofthe level of syn-
DPM
was then rinsed with 1% NaCl and the rinse combined thesis and are best presented as incorporated into
MF
with the homogenate. The homogenate (or the culture (Tobe el al.. 1989).
medium, see next section) was added to acetonitrile and
sufficient saline to give a final acetonitrile:water ratio of Histology
5:4. Ethylfarnesoate(5 or 10 ng)wasaddedasan internal Whole MOs were fixed in modified Karnovsky's(1965)
standard, and the samples were extracted twice with hex- fixative, which consists of 2.7% glutaraldehyde and 2%
ane. MF present in these pooled hexane extracts was de- formaldehyde buffered with 0.1 Msodium cacodylate(pH
termined by npHPLC. Briefly, this method involves sep- 7.3) and adjusted to an osmolality of 835 milliosmoles
arating the hexane extract on a silica column (5 pm, with 10% sucrose. After fixation, the tissue was washed
4.6 mm X 25 cm)with 1.0% diethyl etherin hexane. Ma- with cacodylate buffer, dehydrated in ethanol. and
terial appearing in the same elution volume as 2E,6E embedded at 5C in glycol methacrylate by the method
methylfarnesoatewasquantified byabsorptionat220 nm. ofButler(1984). Sectionswere cut dry at 2 nm thickness
This value was adjusted for recovery by comparison to with machine-made Ralph-Bennett knives(Butler, 1979)
the internal standard. This method has been validated by and stained with either Heidenhain's iron hematoxylin
gas chromatography/mass spectroscopy and has a detec- or Lee's stain (Bennett el al., 1976).
tion limit of 0.1 ng/injection (Borst and Tsukimura,
1991). Results
Measurement ofMFsynthesis Morphology
The synthesis ofMF in vitro was measured in sections The MO has a fan-shaped appearance with a broad
or pieces of MOs incubated at room temperature with fan-folded edge at one end and a narrow smooth (un-
MI PRODUCTION IN THE LOBSTER MO 11
folded) edge at the other end (see Fig. 1 ). //; \//, MOs 250
are oriented with the fan-folded edge posterior and dorsal;
this edge is the major site ofattachment ofthe gland to
the apodeme of the mandibular abductor. The narrow 200
MO
edge ofthe is anterior and ventral. Macroscopic ex-
amination ofseveral MOs suggested that they have two
150
morphological regions. The fan-folded edge ofthe gland
(approximately 25% ofthe glandular area) isthinnerand
MO
hasalight yellow color. The rest ofthe isthickerand 100
dark beige or tan. MOs from animals that had been eye-
stalk-ablated 14 days earlier had a largerand thicker fan-
folded region. 50
MF
content anddistrihution
MF content was measured in freshly dissected whole
MF 50 100 150 200 250
glands. content varied markedly (from 3.2 to
196.8 ng/MO) between animals, with an average content Right MO (ng MF)
of55.5 (12.7. SEM) ng/MO (n = 16). In individual an-
imals, the MF content=s ofthe left and right MOs were leftFiMgOusre.2W.holSeymMmOetsrwyeorfemeextthraycltefdarannesdoathteei(rMMFF)ccoonntteennttidnetreigrhmtiannedd
strongly correlated (r 0.977; Fig. 2). by normal phase high performance liquid chromatography (npHPLC).
MOs from eyestalk-intact and eyestalk-ablated male EachpointindicatestheMFcontentoftheleftandrightMOsofasingle
lobsterswere analyzed to determine regional content and animal(n = 16).Thelinerepresentsthelinearregression;r=0.977and
=
synthesis of MF. MOs from these animals were divided theslope .978.
into four sections ofapproximately equal width (Fig. 1).
For MOs from eyestalk-intact animals, division of the sectedglandswas43.1 (7.7, SEM) ng/MO. Most(95.3%
gland into four sections placed the fan-folded region en- 3.3, SEM)oftheMFineachglandwasfound in section
tirelyin section I. For MOsfrom eyestalk-ablated animals, I, with the remainder(4.7% 3.3, SEM) in section II. No
enlargement ofthe fan-folded region resulted in some of MF was detected in sections III and IV.
MF
this region being included in section II. Although the distribution of in freshly dissected
In every lobster studied (n = 16), MFMcoOntent was lo- MO suggested that MF was producMeFd in the fan-folded
calized in the fan-folded region of the (section I). region in eyestalk-intact animals, might have been
Figures 3 and 4 showthedistribution ofMFin MOstaken produced elsewhere in the MO and then sequestered in
from twogroupsofanimals(eyestalk-intactand eyestalk- the fan-folded region. Therefore, the contralateral MOs
ablated. respectively)that wereanalyzed atthesame time from the animals shown in Figure 3A were divided into
and under similar conditions. Figure 3A shows the dis- sections and incubated separately in medium containing
tribution ofMF in MOs from the eyestalk-intact animals 3H-me/2j>/]methionine. Afterincubation,theMFcontent
(n = 4). One MO from each animal wasanalyzed for MF o[feach section and the MF released into the incubation
MF
immediately after dissection. The total content (the mediumweredeterminedby npHPLC. The incorporation
sum ofthe MF found in sections I-IV) ofthe freshly dis- ofradiolabeled methionine into MFwasalso determined
in these samples.
MF MO
The total content ofthese incubated sections
was 170.7 (36.7 SEM) ng/MO. Of this amount, 7.8%
(0.9, SEM) was released into the medium. Because the
MF content ofpaired MOs from eyestalk-intact animals
MF
is similar (Fig. 1). the amount of produced during
MF
incubation wasdetermined by subtractingthe content
of the unincubated contralateral glands (43.1 ng/MO).
Thus, MF production during the 2-h incubation was ap-
IV proximately 128 ng/gland. Most (90.8% 4.7, SEM) of
the MF contained in each incubated MO was found in
Figure I. Drawingofthelobstermandibularorgan (MO). In some section I, though small amountswere present in the other
ebxypetrhiemheonrtisz,ongtlaalnddsawsehreeddliivneisd.edEaicnthostehcetifoonurwsaescttihoenns(eIx-tIrVa)ctienddiocratiend- threesections(Fig. 3A). Thus, MF production duringthe
cubated in vilrn. The fan-foldededge ofthe gland isin section I. incubation was largely restricted to the fan-folded region.
12 D. W. BORST ET AL
MF
100 The production ofradiolabeled wasstudied further
MO
by dividing the four sections from two animals into
-D 80 IFnrcesuhblayt-eddisMsOected MO sweivtehral[3H(-3/7o7r?76/)n'p/i]emceetsh.ioEnaicnhe painedceMwFasstyhnetMnheFsiinscudbeatteerd-
mined. More than 99%. ofthe radiolabeled was pro-
O 60 duced by piecesobtained from section I. Every piece pro-
duced MF, and those from the middle ofthissection pro-
40 duced the largest amounts (data not shown).
5 The distribution ofMF was also determined in freshly
dissected MOs from eyestalk-ablated animals(n = 4). The
20 total MFcontent ofthese glands was 5,627 (730, SEM)
MF
ng/gland, about 130-fold greater that the content of
MOsfrom thegroupofeyestalk-intactanimals(P< .0001;
/-test). As in the intact animals, the MF content of MOs
III IV from the eyestalk-ablated animals was localized to the
MO
100 fan-folded region ofthe (Fig. 4). Most (74.3% 9.2.
MF
SEM) ofthe in each glandwas found in section I; but
B
a substantial amount (20.3% 9.0, SEM) was found in
80 % Total DPM section II duetotheenlargement ofthe fan-folded region.
The other two sections contained only small amounts of
60 MF(<4%. each).
40
Light microscopy showed that the two regions ofthe
20 MO contain different cell types. Two cell types(A and B)
are found in the fan-folded region ofthe gland. A single
cell type (C) predominates in the rest ofthe gland. The
A cells are a minor cellular constituent ofthe fan-folded
II III IV region and were not alwaysseen. When present, theywere
MO Section found along the outer edge ofthe fan-folded region and
Figure 3. Regional distribution and synthesis ofMF in MOs trom
eyestalk-mtactlobsters. Both MOsfromeachanimal(n = 4)weredivided
into four sections as in Figure I. (A) MF content: Sections ofthe left 100
MO were analyzed for MF content by npHPLC. The MF content of
MF
eachsection isexpressed asapercentageofthe total contentot the
MO (% Total MFContent). No MFwasdetectable in sections III and 80
IV. Sectionsofthe right gland were incubated individually for 2 hours
inHomanissalinesupplementedwith ['H-wi7/n7]methionine.The MF C
contentofeachincubatedsectionanditsculturemediumwasMaFlsoquan- Oo 60
tifiedbynpHPLC"andisdisplayedasapercentageofthetotal content
ofthe incubated MO. (B) Radiolabel incorporation: The radiolabeled uS.
MFintheMOsectionsincubatedabovewascalculated fromtheamount 40
MF
ofradioactivitypresent inthe fractionselutingwith duringnpHPLC. "a
ResultsareexpressedasapercentageofthetotalradioactiveMFproduced o*
bythegland(% Total DPM). 20
This conclusion was confirmed by analyzing the in-
MF
cionrcpuobraatteidonMOof s[e'cHt-i;omnvs/!(Fri/g].me3tB)h.ioTnhineetoitnatlosynthebsyisthoef MIIO SectioIInI IV
rwSoafaEdstMiho)relealrDbeaePadlMsiee/oddlgaMlibnaFetnlodebdt.yhMteOhFfmeeMtfdhoOiisuunsmade.mcoitMnuioonenstast,cwh(a19sM45.6O.367%,%w6a(0s30.3(i1.n6,.s1e7SSc,EEt7i6MMo0))n, sMleobcFFsttiiceogronussnr..teeTE4nh.atecohlfRefesetegacicMtohiOnoassnlecwfdtariissootnmraiinesbaauelctxyhipzoraneendosisffmeoMadrlFaMs(inFna=MpceoO4rn)sctewefnentrrtaoegmbedyeioyvnfeipsdttHehadePlLkti-onCtat.abollafTMtoheuFedr
I,though small amountswerefound in theothersections. contentofthe MO(% Total MFContent).
MF PRODUCTION IN THE LOBSTI-R MO 13
were arranged into cords. Ofthe three cell types, the A MO in vivo (Tsukimura and Borst, 1992). Overall, these
cellsarethesmallest and have the highest ratioofnuclear observations suggest that the mechanisms responsible for
MF
tocytoplasmic material (seeFig. 5). Thesecellsfrequently releasing are impaired duringthe //; vitroincubation.
contain mitotic figures. Their nuclei areoval orirregularly We have previously shown that the percentage of MF
shaped. The A cells have no distinguishing cytoplasmic released in vitroincreases when MOs from Libinia etnar-
features and may be relatively undifferentiated. ginata were incubated in culture medium supplemented
The B cells are the predominant cell type in the fan- with hemolymph. Thus, the hemolymph ofthis crab ap-
MF
folded region, extending from the interior ofthe gland to pearsto contain a factorthat facilitates release (Borst
the A cells, when the latter are present. The B cells are el ul., 1990). Lobster MOs may require a similar factor
larger than the A cells and have smaller nuclear-to-cyto- for MF release.
plasmic ratios, reflectingan increase incytoplasmic mass. Regional production ofMF was also observed in MOs
Their nuclei are round oroval, and theircytoplasm con- from eyestalk-ablated animals. Eyestalk ablation causes
MO
tains many small vacuoles (Fig. 6). Situated between the hypertrophy ofthe in several crustaceans (Byard, et
A cells and the B cells are cells with characteristics inter- ai. 1975; Hinsch, 1977; Le Roux, 1983) as well as an
mediate to those ofthe A and B cells; they may be im- increase in MF synthesis by MOs (Laufer et til.. 1987).
mature B cells. We have previously shown that eyestalk ablation has an
TheCcellsare verylargeand havethesmallest nuclear- acute effect on the MF content oflobster MOs, causing
MF
to-cytoplasmic ratio (Fig. 7). Their nuclei are spherical, a 13-fold increase in content 2 days after eyestalk
and their cytoplasm contains several distinct cytological ablation (Tsukimura and Borst, 1992). Thiseffect iscon-
MF
features, including large vacuolar-like structures. In pre- firmed and extended in this study, in which the con-
MO
liminary electron micrographs (not shown) these struc- tent ofthe increased 130-fold in animals that were
tures contain large masses ofsmooth endoplasmic retic- eyestalkablated for2 weeks. Nevertheless, the increase in
ulum. The cytoplasm ofthe C cells also contains many MFcontent did notaffect thedistribution ofMFin MOs.
MF
mitochondria, but relatively few small vacuoles. Thus, the rest ofthe gland does not represent reserve
production capacity that can be activated when thegland
Discussion isstimulated. These observationsdo not rule outthe pos-
sible involvement ofother glandular regions in the syn-
MO MF
The gross morphology ofthe lobster suggests that thesis of precursors such as farnesoic acid.
the fan-folded edge is distinct from the rest ofthe gland. Analysisofpaired MOs from individual lobstersshowed
This wasconfirmed by the studies reported in this paper, that theglandscontain similaramountsofMF. Thiscon-
which demonstrated that these two regions differ in both firms and extends a previous study, in which the mean
their biochemical and histological characteristics. MFcontents ofright MOsand left MOswere statistically
Biochemical studies showed that MF in unincubated similar (Tsukimura and Borst, 1992). The symmetrical
MOs was localized in the fan-folded region of the MO. MF content ofpaired MOs was a critical prerequisite for
MF MF
The possibility that this region is the site of synthesis measuring production during incubation.
was tested by determining the production of MF by in- Thesimilar MFcontent ofpaired lobster MOssuggests
dividual MO sections in vitro. The MFmeasured in these that these glands produce MF at similar rates //; vivo. In
sectionswasfoundlargelyinthefan-foldregion, indicating contrast, the in vitro biosynthesis ofMF by paired MOs
that MF synthesis was occurring primarily in this region. from the mud crab Scylla serrata and the crayfish Pro-
The production of radiolabeled MF was also highest in cambarus clarkii is asymmetric (Tobe et a/.. 1989; Ding
thisregion. Taken together, thesestudiesdemonstratethat and Tobe, 1991). In addition, asymmetric biosynthesisof
MFproduction isan important function ofthefan-folded JH in vitro has also been observed in the corpora allata
region ofthe MO. from several insect species (Tobe and Stay, 1985). How-
The total MF content of the incubated MO sections ever, such comparisons are difficult to interpret. The re-
MF
was fourfold greaterthan that ofthe unincubated contra- lationship of in vitro synthesis to the activity ofthe
lateral MO.Thus,theproductionofMFbythe MOduring MO //; vivois not clear, because tissues incubated in vitro
incubation wassubstantial [63 ng(256 pmole) per hour]. are no longer controlled by endogenous regulatory sys-
This level fallswithin the rangeofsynthetic ratesobserved tems. For the lobster, this is particularly significant, be-
previouMslFy for other specieMsO(Sagi et a/., 1991). About 8% cause eMyeOstalk factors can rapidly affect MF production
ofthe present in the was released during the 2- by the (Tsukimura and Borst, 1992).
h incubation, confirming reports by others (Tobe et ai. Our histological studies also show two distinct regions
M19O89;isDlionwg.aTnhdisTolboew, l1e9v9e1l)otfhaMtFMFrelreealseeasienbyvitirnocusbtaatnedds cienllthteypelso.bsTthereMphOy.sicTahlereflaant-ifoonlsdheidp orfegtihoenseccoenltlsa,inasswtewlol
MF
in contrast to the apparent rapid release of by the as the presence of morphologically graded transitional
14 D. W. BORST /;/' I/
6
"
'
A
;
Figure5. Light micrograph ofAcellsfoundin fan-folded region ofthe MO. Notearrangementofthese
cells into cords. The cells have irregularly shaped, small nuclei and a homogeneous cytoplasm. Mitotic
figuresare present. Scale = 5^m.
Ml PRODUCTION IN THE LOBSTER MO 15
forms, suggests that A cells provide the precursor cells TexasChristian University. Thisstudy isapublication of
thatdifferentiateintoBcells. Basedontheirmorphological the IntensiveCommercial AquacultureFacility ofIllinois
characteristics and location within the fan-folded region, State University. We thank Dr. J. K. Butler for his help
the B cells are the most likely site ofMF synthesis. in these studies, and express our appreciation for the
The rest ofthe gland contains predominantly C cells, helpful comments from two anonymous reviewers and
whose function and relationshiptotheothertwocell types the editors ofthisjournal.
are unknown. Preliminary ultrastructural studies suggest
that these cells are active metabolically and may be in- Literature Cited
We
volved in the synthesis oflipids orsteroids. speculate
thatthey maybethesitesofthe progesterone metabolism Aoto,T., V. KMMHUIMIn, and S. Ilisano. 1974. Histological and ultra-
previously reported in MOs from female lobsters (Couch structural studies on the V organ and the mandihular organ ofthe
oeluruik.no1w9l87e;dgTesuMkOimsurfar,om1m98a8l)e.lHoobswteevresrh,avteo ntehevebresbteeonf 2frer9le5as-th3iwo0an8ts.erwiptrhawnth.ePamloaletniinngncpyactltec.id.e/nxF.acw.ithScsi.pecHioalkkreafiedroencUenitvo.th1e9i:r
investigated for the presence ofthis steroid. In any case, Bennett, H. S., A. D. Wyrick, S. W. Lee, and H. J. McNeil, Jr.
the lobster MO may have several products, so the phys- 1976. Scienceandart in preparingtissuesembedded in plasticfor
iological importance ofthis tissue may be complex. light microscopy with special reference toglycol methacrylate. glass
The MhisOtological and biochemical complexity of the Borsktn,ivDe.sVaV.n,dasnidmIpIl.eLsatuafienrs.. S1l9a9i0n. TcMchentohly.l5f1a:rn7e1s-o9a7te..aJH-likecom-
lobster was unexpected, partly because ofthe simi- poundincrustaceans. Pp.35-60inRecentAdvancesinComparative
larity between the MOs ofother crustaceans and the CA ArthropodMorphology. Physiology, andDevelopment. A. F. Gupta,
of insects. In insects, the CA contains only one type of ed. Rutgers University Press, New Brunswick. NJ.
go1lb9as7ne4dr;uvlaBatyriaocrnedlsleo(tfTaMot.bO.es1a9nf7dr5;oSmtDaeoyt.mhee1u9rs8c5yr),.usL1ti9ak7ce5ew;ainsHsei,(nAscoyctthoolaoenglidtciala.ll. BBoorrssntItoe,,gsroDDaa..pthe\\yVV...l,,e.v/aeH.ln.CsdhLirBano.umfhTaeestrmu,okogMirlm..yumr5Lp4aa5h.n:db1a7y9u19,-1h7.iE8.g.hS-Q.puaeCnrhtfaionfrgim,caantWci.eonAl.ioqfuHimedrettczhh,yrloF.mfaaCr-.-
Hajj, 1975; Hinsch, 1977; Buchholz and Adelung, 1980) Baker, and D. A. Schoolcy. 1987. Methyl farnesoate and its role
also suggest that the glands are relatively homogeneous. in crustacean reproduction and development. Insect Biochem. 17:
MOsfrom adult female spidercrabs, L. emarginata, have 1123-1128.
two cell types that differ in their staining properties, but Borsmt,etDh.ylW.f,arCn.eGs.oaBtueerrkeelteta,seanfdroBm.Tmsaunkdiimbuurlaa.r1o9r9g0.ansF(aMctOosr)saifnfevcittirno.g
have similar ultrastructural characteristics. In addition, Am. Zool. 30: 29a.
there is no indication that these cells were regionally dis- Buchholz,C.,and D. Adelung. 1980. Theultrastructuralbasisofsteroid
tributed in the MO ofthis species (Hinsch, 1981). Like- production in the y-organ and the mandibular organ ofthe crabs
wise, we found no evidence for regional distribution of //cmwu/'wn niuliis(Dana)and CannnismaenasL. CellTiss. Res.
MF synthesis in MOs from L. emarginata (Tsukimura. Butl2e0r,6:J.83K-.9149.79. Methodsforimprovedlightmicroscopemicrotomy.
Martin, and Borst, unpub. data). Slain Techno!. 54: 53-69.
MO
Ourstudiesshowthat the lobster is more complex Butler, J. K. 1984. Improved methods for molding, facing, and sec-
than the MOs ofother species, containing at least three tioningofglycol methacrylateembeddedtissueblocks.Slain Teclmol
cleolblstteyrpeMsOloacaplpiezaerdsitnotbweounairqeause,ofantdhemgalyanrde.prTehMsueFsn,t tthhee Byar5od9r,:ga3En.15oH-f.3,t2h1Re..loRb.stSehri,velrlso,maanrduxD.ameEr.icAaiikneins..1C9e7l5l.TissT.heResm.an1d6i2b:ul1a3r-
fusion oftwo tissues, one ofwhich synthesizes and TT
the otherofwhich produces one or more other products. Gassier, P. 1979. Thecorporaallataofinsects. //;/. Rev. Cylol. 57: I-
73.
Acknowledgments Couch, E. F., N. llagino, and J. \V. Lee. 1987. i ranges in estradiol
andprogesteroneimmunoreactivity intissuesofthelobsterHomarus
This research was supported by grantsto D.W.B. from umericanit*. w-ithdevelopingandimmatureovaries. Comp. Biochem.
the National Science Foundation (DCB-8919833) and the rhysiol. 87A: 765-770.
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Figure6. LightmicrographofBcellsinthefan-foldedregion.Thenucleiofthesecellsareovalorround
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Figure 7. Light micrograph ofCcells in the unfolded region. Note the largespherical nuclei and large
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