Table Of ContentReference: Bio!. Bull. 195: 112-114. (October. 1998)
Effect of Methyl Farnesoate on Late Larval
Development and Metamorphosis in the Prawn
Macrobrachium
rosenbergii (Decapoda,
A
Palaemonidae): Juvenoid-like Effect?
URI ABDU'. PETER TAKAC2 HANS LAUFER3 AND AMIR SAGI1 *
, ,
1 Department ofLife Sciences, Ben-Gnrion University ofthe Negev, P. O. Box 653,
Beer-Sheva 84105, Israel; 2Institute ofZoology, Slovak Academy ofSciences, Bratislava 84206,
Slovak Republic; ami 'Department ofMolecular and Cell Biology, University ofConnecticut,
Storrs, Connecticut 06269-3125
Abstract. Methyl farnesoate (ME), the unepoxidated al., 1985), but its physiological significance is not clear.
formofinsectjuvenile hormone III. wasdetected in larvae The first report of the presence of MF in Crustacea was
of the freshwater prawn Macrobrachium rosenbergii, that of Laufer et al. ( 1987), who demonstrated that MF
which metamorphose to post-larvae following 1 1 larval is secreted by the mandibular organ of the spider crab
stages. The possible role of ME as a morphogen was Libiniu emarginata and is found in its hemolymph. Since
studied by administering the compound toM. rosenbergii then, MFhasbeen found in avariety ofcrustaceans (Borst
larvae via an Anemia vector. Higher ME levels caused i-tat.. 1987; Laufer and Borst, 1988; Landau etal., 1989),
earlier retardation of late larval growth, and the highest including the freshwater prawn Macrobrachium rosen-
ME
dose retarded larval development. Furthermore. sig- bergii (Sagi et al., 1991 ).
nificantly affected the patterns of metamorphosis and the JHs are involved in the regulation ofa substantial num-
appearance of intermediate individuals exhibiting both berofinsect functions, including development, reproduc-
larval and post-larval morphology and behavior. Three tion, and behavior. JHs are probably best known for their
intermediate types weredefined, two ofwhich were found effect on the growth and differentiation of insect epider-
only at the MF-treated groups and one that was exclusive mal cells. When JHs are present during larval instars, the
to the higher dose treatments. The relative abundance of cuticle that is secreted has larval characteristics; in the
intermediate specimens increased from 2% in the control absence of JHs. the cuticle exhibits pupal characteristics
to 32% in the high MEconcentration, which suggests that (see Laufer and Borst, 1983, for a review). Exogenous
ME
has ajuvenoid-like effect in this decapod crustacean. application of JHs may cause the appearance of various
intermediate forms, and in some cases, of supernumerary
Introduction larvae, as a result of differences in the growth rates of
Juvenile hormones (JHs) a family ofepoxidated ses- various parts of the body (allometric growth) (reviewed
quiterpenoids act as growth regulators in insects (Wig- in Sehnal, 1983). It has been suggested that exogenous
glesworth, 1970). Methyl farnesoate (ME), the unepoxi- JH applied during a critical period may function at the
dated form of JH III, has been detected in some insect cellular level, resulting in mosaic animals with character-
species (Slama. 1971; Lanzrein el ai. 1984; Bruning et istics of two different stages (see Riddiford, 1994, for a
review). Thus, it is likely that the effect of a particular
juvenoid on insect development is determined by the de-
R*eAcuetihvoerd 6toNowvheommbercor1r9e9s7p;oancdceenpcteedshIoIIulJdulyhe19a98d.dressed. E-mail: velopmental timing and the effective concentration ofthe
juvenoid (Slama. 1995).
[email protected]
Ahhreviatiims: MF, methyl farnesoate; JH,juvenile hormone. The regulation oflarval development and metamorpho-
112
MF AND METAMORPHOSIS IN MACROBRACH1UM 113
sis in crustaceans is not fully understood. In cyprids of washed with4% NaCl and acetonitrile, and extracted with
barnacles, JH I and JH analogs caused premature meta- hexane. The hexane fraction was evaporated by means of
morphosis without attachment of the cyprid to the sub- a rotary evaporator. The dry matter was dissolved in hex-
strate (Gomez et cil., 1973; Ramenofsky et al., 1974; aneandpassedthrough aSep-Paksilicacartridge (Waters,
Tighe-Ford, 1977). In decapods, JH analogs retarded the Milford, MA), and the cartridge was rinsed with methy-
development of larvae ofthe mud crab Rhithropanopeus lene chloride (Abdu etal., 1998). The methylene chloride
htirrixii (Christiansen et al., 1977a, b) and the growth of was evaporated, and the residue was dissolved in 100 /j\
early larval stages and post-larvae ofthe estuarine shrimp of hexane, loaded into a Beckman HPLC and analyzed
Palaemonetes pugio. In contrast, growth was enhanced according to Sagi etal. ( 1991 ). The nonbiological isomer,
in the premetamorphic stages of P. pugio (McKenney cis-trans MF, was used as the internal standard, and the
and Celestial, 1993). Treatment of Homants americanus retention times were compared to an external standard
MF
larvae with JH III increased the duration of larval devel- containing both the biological (all-trans ) and nonbio-
MF
opment and influenced the size of the carapace and ap- logical isomers. The putative trans-trans peaks of
pendages (Hertz and Chang. 1986). Injections ofJH I into larvae at stages 4, 6, 9, 10, and 1 1 were collected from
H. americanus larvae gave rise to a number ofintermedi- the HPLC and loaded onto a Hewlett Packard gas chro-
ate stages (Charmantier et al.. 1988). matograph with a 30-rn cross-linked and surface-bound
MF is the only JH-like compound found thus far in dimethylpolysiloxane column (0.5 min to 70C. 70-
crustaceans. A few studies have targeted its regulatory 280C. 20C/min) connected to a mass spectrometer.
role in development and metamorphosis in decapods. Ap-
plication of MF to H. americanus larvae prolonged meta- Effect ofmethylfarnesoate on late larval growth and
morphosis by a small but significant amount of time development
(Borst et al.. 1987). In M. rosenbergii, MF retarded Three experimental groups, each containing 15 M. ro-
growth and development in larval stages 5 to 9 (Abdu et senbergii stage-9 larvae, were fed with Anemia enriched
acmtiaae.sbnIeontl1fo9saou9rnos8dmM).e.imnesrcetorcsautemssnotbra(ecpSrehgnaioynisds,eirswthhaaeirncedphrsCoeicxhmheiailsnbasgire,tstso1oa9tf8"h6olc)aso.revmaSimlnuochdnheevmtieisylmpoteeph"--e cawwSlaiiE.tttLe(hhsC1.O09t.T9)h28h.e1)e..EevA0axe.cp3hc5eiho,crnlittoermrroeelna0att.glam5orle9noneust/pyjgsw(wteaMatesFshmy/lfpcmeeoldnrasfwliaoicscrtotcmhheooedArlddnoifeinanmgn1i2dttahodraSeeArenUbkrPdirpeEculphRalesie-d--t
of larval development (Sollaud. 1923). encompassing 11 tic containers (300 ml) that were constantly aerated. The
satnadgeSso,of,ol1l9o69w)e.dLbayrvmaeltadmeovreplhoopsmiesntinitnoMp.ostr-olsaernvbaeerg(iUinios twaaitneerdwaats27replac1eCd.daTihley,laanrdvaethweetreempfeerdataudreliwbiatsummawiint-h
characterized by significant growth of the carapace from Anemia nauplii that were enriched daily with MF. Five
stage 1 through stage 11. During metamorphosis (from larvae perreplicate were randomly sampledtwice a week;
stage 1 1 to post-larva), growth is allometric, with in- larval stage wasdetermined andthe length ofthe carapace
creases in size being limited to certain parts of the body was measured with an ocularmicrometer; the larvae were
(excluding the carapace) (Abdu. 1996). then returned to the growth chamber. In the calculation
In this study, M. rosenberMgiFi was used as a model spe- of the average larval stage for each sampling, all post-
cmieenstfoarnsdtumdeytianmgorthpehorsoliesooff crusitnatcheeanlsa.teMlaFrvawlasdevienldoepe-d i1n1disvtiadgueasls)(inwcelruedidnegsibgontahtepdosats-la1r2.vaeThaendavienrtaegremeldairavtael
odeftemcotrepdhoinlotghieclaalrvafee,ataunrdesitsduerfifencgt omnettahmeormapnhiofseisstatwiaosn bsteatgweeeanndtrtehaetmeanvtesr,agaendcatrhaepascteatilsteincgatlhswigenrieficcaonmcpeawraesd
studied. ANOVA
tested using one-way followed by the least sig-
nificant difference (LSD) test P = 0.05. The experiment
Materials and Methods
wasrepeated three times with similarresults; each experi-
Detection ofmethylfarnesoate in Macrobrachium ment contained 180 larvae.
rosenbergii lan-ae
Assessment ofmetamorphosis in Macrobrachium
M. rosenbergii larvae were hatched and reared ac-
rosenbergii
cording to Daniels etal. (1992). Larval stages were deter-
mined according to Uno and Soo (1969). To detect the Among the many changes occurring during metamor-
presence of MF, about 1 g of larvae at each ofthe stages phosis (Ling, 1969; Uno and Soo, 1969), the following
4, 6, 9, 10, and 1 1, and post-larvae were collected and six predominant changes were selected to distinguish be-
homogenized in 2 ml of4% NaCl and 5 ml ofacetonitrile. tween larvae and post-larvae of M. rosenbergii in this
The homogenates were filtered through a-cellulose. study; ( 1) In larvae, the abdomen is bent such that the
114 U. ABDU ET AL.
cis-transMF the pereiopods. During the metamorphic process these
4.00 -I 4.88 long exopodites degenerate and become reduced to knobs
at the postmetamorphic molt (Ling, 1969). (3) In stage
1 1 larvae, the endopodite of the second pereiopod has
three segments, the second and third segments being at
3,00- an angle to the first, which is not fully developed. In
contrast, in post-larvae there are four segments, all of
which are straight, and the first segment is fully devel-
oped, as in the adult. (4) The carapace median tooth is
evident from larval stage 4 onward, but disappears during
metamorphosis and is absent in the post-larva. (5) In stage
I 1 larvae, the entire dorsal margin of the rostrum is
equipped with numerous small teeth, but no ventral teeth
1.00 are present, whereas the rostrum of the post-larva has
I 1 large dorsal teeth and 5 ventral teeth. (6) Larvae are
planktonic. swimming tail up. and in most cases tail first,
ventral side up; during metamorphosis, they settle to the
5.00 5.50 6.00 bottom. Post-larvae are benthic, swimming and behaving
as do the juveniles.
Retention time(min ) The relative abundance of the different intermediate
Figure 1. HPLC detection ofmethyl farnesoate in Macrdbnicliiuin types were compared between treatments, and the statisti-
rosenbergii larvae at stage 4. Cis-trans methyl farnesoate was used as cal significance was tested using the Mann-Whitney U
the internal standard.
test.
Results
pleopods and the walking legs are not in the same plane, Presence ofmethylfarnesoate in Macrobrachium
whereas in post-larvae, the abdomen is straight, with the rosenbergii larvae
pleopods and the walking legs in the same plane. (2) MF was found in hexane extracts of M. rosenbergii
Larvae swim mainly by beating the long exopodites of larvae at stages 4, 6, 9, 10, and 1 1, and in post-larvae. A
50,000 1 69
<Uu
c
03
25,000 -
3
-C.
114
121
250
n"1 lUillU ililil -U4VA- 'M ,"/l,ii Jm -^t+i
40 80 20 160 200 240
m/z
Figure 2. Mass spectrum of methyl farnesoate extracted from Macrobrachium rosenbtrgii larvae.
Methyl farnesoate peaks were collected from HPLC separation of larvae extract and analyzed by GC-MS.
MF AND METAMORPHOSIS IN MACROBRACHIUM 115
pace length was observed in the late larval stages of M.
rosenbergii exposed to higher doses ofMF (Fig. 3). The
control group and the larvae fed on Anemia that were
1500 enriched with the lowest concentration of MF exhibited
the fastest growth. Growth retardation in larvae fed on
Anemia enriched with the median dose of MF was sig-
E:! nificant (P < 0.001 ) only on day 12. Growth of larvae
fed on Anemia that were enriched with the highest con-
Ta,. centration of MF was markedly slower than that of the
a <
control group, the difference being significant (P
0.001) from day 5 onward; after that time, the "high-
MF
dose" group almost ceased growing. also retarded
1200 larval development, as manifested by larval stage (Fig.
4). The control group and the larvae fed on Anemia
Days MF
enriched with the lowest concentration of developed
rapidly, reaching an average larval stage above 10 after
5 days and an average larval stage of above 1 1 at the
MF(ug/ml)
Figure 3. Average carapace length ofMacrobrachium roxenbergii
larvae during 12 days of feeding on Anemia enriched with different
concentrationsofmethyl farnesoate. Each barrepresents mean SE(n
= 151. The results presented are taken from one representative experi-
ment out of the three identical experiments performed. Using the Ar-
lemia vector, approximately 0.0325% ofthe MF added to the Anemia
enrichment medium (referredtoas MF(^g/ml))was found inM. rosen-
bergiilarvafeeding for8hontheenrichedAnemia (Abduelal., 1498).
This suggests that the level of MF in the present study did not exceed
0.191 ng MF per larva exposed to the highest dose and 0.068 ng MF
per larva exposed to the lowest dose. Since the average weight of a
larva is about 0.002g, these levels could be expressed as 34-95.5 ng
MF/g. The calculated level for the low MFexposure is in the range of
physiological level found in M. rosenbergii larvae (up to 25 ng/g, data
not shownl and adults (up to 40ng/ml, Sagi et al., 1991). Growth
retardation in larvae fed on Anemia enriched with the median dose of
MF was significant (P < 0.001) only on day 12. Growth oflarvae fed
on Anemia that were enriched with the highest concentration of MF 5 Days
wasmarkedlyslowerthanthatofthecontrolgroup,thedifferencebeing
significant (P < 0.001) from day 5 onward.
representative HPLC chromatogram of one such hexane
extract of larvae shows the presence ofthe biological all-
MF(|ag/ml)
trans MF, identified by comparing its retention time with
that of the external standard (data not shown) and that of Figure4. Average larval stageofMacrobrachium rosenbergiidur-
the internal standard (Fig. 1). Gas chromatography-mass ing 12daysoffeedingonAnemiaenrichedwithdifferentconcentrations
spectrometry of the putative peaks from larval stages 4, of methyl farnesoate. Each bar represents mean SE (n = 15). The
6, 9, 10, and 1 1 showed atypical methyl farnesoate profile rtehseultthsrepereisdeenntteidcalareextpaekreinmefnrtosmpoenreforremperde.seMntFati(v/ejge/xmpl)eriremfeenrts otuottohef
exhibiting selected ion monitoring at m/z 69, 1 14, and amountofMFaddedtothemediuminwhichtheAnemiawereincubated
121 (Fig. 2). prior to being provided as food to M. rosenbergii larvae. In the larvae
fed onAnemia enriched with the median dose ofMF, furtherdevelop-
Effect ofmethyl farnesoate on late larval growth and mentwassignificantly retardeduptothe5thday(P< 0.001,vs. control
group), but not from day 5 to day 10. After day 10, developmental
development progress was again markedly slower, and on day 12 the average stage
minenStuthrevgitrrvoeaualptsewda(gs3r5os%uimpisilnafcrroonitnmrottlhh,ee aclnoondwtr4to7ol%t,ahne4dh1ia%gl,lhtaMhneFdt4rceo6ant%-- ot(MhfPFet<hcisoshn0tto.rr0woe0ela1dt)g.mresoinLgutanprig,vfraioscetuaapnfrtetlwdiyangsosnlosoniAgwrnedtirafeyimdciea5anvte(ellPynorbp<iemcheh0ine.ndt0d0w1ttoi)h;taahtdtohtvifhasetnhechefeifdgcehocnetststrtlaoaglsdetosgesdrteohtuaiolpnfl
centration, respectively). An earlier retardation of cara- the end ofthe experiment.
116 U. ABDU ET AL.
end of the experiment (after 12 days). In the larvae fed Effect of metlivl farnesoate on metamorphosis
onArtemict enriched with the mediandose ofMF, further
development was significantly retarded up to the 5th day Metamorphosis which takes place after the 1 1th lar-
(P < 0.001, vs. control group), but not from day 5 to val stage was delayed in a dose-dependent manner in
day 10. After day 10, developmental progress was again larvae treated with MF, but survival was similar to that
markedly slower, and on day 12 the average stage of in the control. In summary, the three experiments showed
this treatment group was significantly behind that ofthe that among the surviving control individuals, 28 (58%)
control group (P < 0.001). Larvae fed on Anemia en- reached the postlarval stage at the end ofthe experiment.
riched with the highest dose ofMF showed significantly In the low MFconcentration 20 (31%) normal post-larvae
MF
slower development to advanced stages than the control were found, and as higher concentrations were used,
group, starting on day 5 (P < 0.001); this effect lasted significantly fewer post-larvae were found only 8
till the end of the experiment, and only a small progres- (14%) and 5 (8%), respectively. In addition to the delay
sion oflarval stages was recorded from that day onward. in metamorphosis, different forms possessing both larval
Larva Post-larva
Intermediate
Larva
Post-larva
Figure 5. Schematic drawing oflarval and postlarval features in a representative intermediate Mucro-
brachium rosenbergii. R- rostrum, MT- median teeth. P- propodus. Ex - exopod ofthesecondpereiopod.
En - endopod ofthe second pereiopod.
MF AND METAMORPHOSIS IN MACROBRACHIUM 117
us U. ABDU ET AL.
bergii (Abdu, 1996: Abdu et <//.. 1998); and finally, the organ-inhibiting hormone (MOIH) from the sinus gland
findings of the present study in which the retardation of in the eyestalk (Liu and Laufer. 1996; Wainwright et al,
metamorphosis and the appearance of intermediate M. 1996), together with the present work, suggest that MF
rosenbergii types were observed after larvae were ex- could be the direct hormonal agent controlling metamor-
posed to exogenous MF. phosisincrustaceansthroughanindirecteffectofeyestalk
In insects, inhibition ofmetamorphosis is primarily due neuropeptides.
MF
to the morphogenetic action ofajuvenoid. This morpho- has recently been described as a "crustaceanjuve-
genetic effect is routinely scored by recording the preser- nile hormone in search of functions" (Homola and
vation of juvenile characters after the metamorphic ecdy- Chang, 1997b). The findings of the present study the
sis. Typically, early treatment of the last-instar larvae delay in larval development and metamorphosis in M.
induces molting into supernumerary larval instars. Later rosenbergii, together with the increase in the abundance
treatment results in a reduced effect appearance of in- of intermediate specimens with the increase in exogenous
MF
termediates between the juvenile and metamorphosed contribute to the debate over the physiological
stages since the various body tissues gradually become function of MF, supporting its possible role as ajuvenile
insensitive to juvenoids (for review see Sehnal, 1983). hormone in the development and metamorphosis ofcrus-
As with insects, the most striking result ofthis study was taceans.
the appearance, in the MF-treated groups, ofintermediate
M. rosenbergii individuals. It is notclearhowour findings Acknowledgments
relate to those in insects whether the different interme-
We thank Ms. Hanna Ben-Galim and Ms. Galit Yehez-
diate individuals in the present study correspond to the
supernumerary larvae or the intermediate forms known kel for their technical assistance, and Ms. Inez Mureinik
in insects. for editorial review ofthe manuscript. A. S. is the incum-
The retardation in larval growth found in this study bent of the Judith and Murray Shusterman Chair for Ca-
may be due to a molt inhibition, as in some insect species reer Development in Physiology.
in which continuous exposure to juvenoids can cause a
permanent molting block (see Sehnal, 1983, forareview). Literature Cited
On the other hand, juvenoids can accelerate the moltMinFg Ahdu, U. 1996. The involvement ofmethyl tarnesoate. administered
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(Chang et til.. 1993); this suggests that in M. rosenbergii rosenbergii. M.Sc. Thesis. Ben-Gurion University of the Negev,
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MF AND METAMORPHOSIS IN MACROBRACHIUM 19
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