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JOURNAL OF THE
WORLD AQUACULTURE SOCIETY
Practical Molt Staging of Penaeus setiferus
and Penaeus stylirostris
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Vol. 18, No. 3
September, 1987
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LORIROBERTSON,
WILLIAMBRAY,JOANNA
AND ADDISON
LEUNG-TRUJILLO
LAWRENCE~
Shrimp Mariculture Project, Texas Agricultural Experiment Station, Texas A&M University
4301 Waldron Road, Corpus Christi, Texas 78418 USA
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Abstract
Molt staging has many applications in penaeid aquaculture as a routine procedure. Using criteria
developed for various crustaceans, a method of rapid molt staging was applied to Penaeus stylirostris
and Penaeus setiferus adults. The most useful characters were the degree of epidermal retraction
from the setal bnses and epidermal retraction from the cuticle coupled with the development of new
setae. The duration of the molt cycle of P. stylirosrris was 11.5 + 1.0 days (N = 5) at 27-29 C. The
early postmolt, late postmolt, intermolt, early premolt and late premolt stages were readily deter-
mined. For P. stylirostris, the duration of postmolt was 27% of the cycle; intermolt, 17%; and
premolt, 56%. The length of the molt cycle of P. setiferus at 27-29 C was 13.6 k 1.0 days ( N =
21) with postmolt occupying 22% of the cycle; intermolt, 19%; and premolt, 59%. Unilaterally
eyestalk enucleated P. setiferus had a significantly shorter molt cycle than intact P. setiferus (11.7 k
1.21 days vs. 13.6 ? 1.02 days, P < 0.05).There were no differences in molt cycle duration between
the sexes, whether enucleated or intact (P > 0.05). A series of illustrations identifies the key
characteristics for determining stage of molt in these penaeids.
Since molting and reproduction are asyn- stages (Drach 1939). Stage determinations
chronous activities (Emmerson 1980; An- are made using various criteria, including
derson et al. 1985), the ability to determine external changes in the color and feel of the
when and for how long the various stages exoskeleton and internal morphological
of the molt cycle occur would be highly use- changes associated with the withdrawal of
ful in penaeid reproduction facilities. Molt the epidermis from the cuticle (apolysis) and
stage determinations of female broodstock the development of new setae (setogenesis).
could be used for: 1) prediction of repro- The morphological changes have been mi-
ductive peaks and thus larval production croscopically observed in pleopods (Schafer
cycles (Browdy and Samocha 1985); 2) for 1968; Longmuir 1983), uropods (Read 1977;
the timing of eyestalk enucleation, since Huner and Colvin 1979) and in histologi-
enucleation during early postmolt causes cally prepared abdominal sections (Smith
mortality (Aquacop 1977,1979) and during and Dall 1985). The existing literature on
late premolt, delays ovarian development molt staging of Penaeus spp. is somewhat
(Crocos and Kerr 1983; Browdy and Sam- difficult to interpret and apply for practical
ocha 1985); and finally, 3) molt staging pond use. The critical role of the molt cycle in
animals could be important for timing of penaeid culture has created a need by aqua-
harvest since the condition of the exoskel- culturists for a rapid, easy method o f dis-
eton influences market value (Apud et al. tinguishing the major molt stages.
1983). This paper presents a method of molt
The molt cycle of crustaceans is divided staging that can be performed in less than
into five major stages with numerous sub- 45 s on live shrimp or on excised uropods.
Using this method, the duration of the var-
ious molt stages was determined for adult
I Corresponding author. Penaeus stylirostris and Penaeus setiferus.
5 ~ \ C'opynght by the World Aquaculture Society 1987
180
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MOLT STAGING OF P. SE7'Il;t'RL'S A N D P. STYLIROSTRIS
Materials and Methods termolt (Stage C), early premolt (Stage Do-
Thirty Penaeus setiferus adults (males = DI),and late premolt (Stage D2-D3). Ecdysis
43 k 7.1 g; females = 57 * 5.8 g) sourced (Stage E), the actual shedding of the exo-
skeleton, occurred in less than one min. Fig.
from the Gulf of Mexico and seven adult
Penaeus stylirostris males (52 k 6.9 g) col- 1 illustrates the succession of changes that
lected from the Pacific coast of Mexico were occurred in the uropods throughout the molt
observed for stage of molt every one to two cycle and identifies the key characters for
days for seven weeks. Both species belong distinguishing stages in P. setiferus and P.
to the subgenus Litopenaeus and have open styhostris. There were no species differ-
thelyca. Each animal was tagged with a ences in the pigment and structural changes
numbered elastic band placed around the which occurred, although the durations of
eyestalk so that individual shrimp could be the stages varied. The pigment retraction
monitored throughout the molt cycle. For and structural changes which occurred were
observation, shrimp were placed singly in a also the same for male, female, enucleated,
restraining device (as described by Olivier and intact shrimp. No external changes in
and Salinger 1985), and a plexiglass plate coloration were evident throughout the molt
(6 x 6 x 0.4 cm) was placed below the cycle. During Stage A, which occurred im-
telson and uropods. The setaelsetal base in- mediately after ecdysis, the pigmented cel-
terface of the uropods was illuminated and lular matrix completely filled the setal bases.
observed under a dissecting microscope at During Stage B, the cellular matrix retracted
10-70 x magnification. The animals were from the setal bases and was easily recog-
staged using two criteria: 1) presence or ab- nized by the clear space present in the bases
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sence of cellular matrix in the setal bases; as retraction occurred. In Stage C , the ma-
and 2) degree of epidermal withdrawal cou- trix was absent from the setal bases and the
pled with the degree of development of new pigment appeared to form an epidermal line
setae. Using these criteria, Read (1977) and at the bases of the setal nodes. When the
Huner and Colvin (1979) previously deter- pigment retracted from the bases of the
mined molt stages in P. indicus and P. sty- nodes, leaving the old cuticle, Stage Do-DI
lirostris, and in P. californiensis, respective- began. During D2-D3, the pigment retracted
ly. To determine the effect of eyestalk further and the development of new setae
enucleation on the molt cycle, five male and was observed.
five female P. setiferus were unilaterally Using the above criteria, live P. setiferus
enucleated during early premolt and ob- and P. stylirostris were rapidly molt staged.
served for a complete molt cycle. During Some mortality occurred in both species, in
the study, seawater temperature ranged from both sexes and in both enucleated and intact
27 to 29 C , salinity ranged from 34 to 41 animals. Thus, complete cycles were not de-
ppt and the photoperiod consisted of 13 h termined for all shrimp initially stocked. The
light and 1 1 h dark. The animals were fed duration of the molt cycle of P. setiferus was
squid and a dry diet two to three times daily. 13.6 f 1.0 days ( N = 2 1) with postmolt (A
T-tests were used to determine differences and B) occupying approximately 22%; in-
in molt cycle duration between treatments. termolt (C), 19%; and premolt (D), 59% of
Differences were considered significant at the cycle, respectively (Table 1). For P. sty-
P < 0.05. lirostris, the length of the molt cycle was
1 1.5 f 1.O days ( N = 5) with postmolt oc-
cupying approximately 27%; intermolt, 17%;
Results
and premolt, 56% of the cycle, respectively
The molt cycle was divided into five stages (Table 1). The molt cycle was significantly
that were distinguished readily: early post- shortened from 13.6 1.02 days in intact
molt (Stage A), late postmolt (Stage B), in- P. setiferus ( N = 2 1) to 1 1.7 k 1.21 days in
182 zyxwvutsrqp
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/
/
/
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/
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seta- zyxwvu
setal base
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cellular matrix
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STAQE A STAQE B
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FIGUREI . Stages of the molt cycle identified by the succession of changes occurring at the setaehetal base
interface of the uropods of adult P. setiferus and P. stylirostris. See the text for concise descriptions of the
various stages. The durations shownfor the stages are those of P. stylirostris maintained at 27-29 C.
unilaterally eyestalk enucleated P. setiferus ration between the sexes, whether enucleat-
(N = 6). Early premolt was the stage most ed or intact.
affected by enucleation, decreasing from four
to two to three days duration. There were Discussion
no significant differences in molt cycle du- For an aquaculturist, the most critical
times of the molt cycle are immediately be-
fore and immediately after ecdysis (i.e., dur-
TABLE
1. Molt cycle duration of adult P. setiferus and ing late premolt and early postmolt). It is
P. stylirostris. during these stages that reproduction ceases,
that stress has its most adverse impact, and
Time in daysa
that the shrimp have lowest market value.
P. set$ P. styli- For this reason, it is unnecessary and im-
Stage erus N rostris N
practical for culturists to accurately sub-
Early postmolt (A) 1 9 1 6 stage their animals to any further degree. A
Late postmolt (B) 2 8 2 6
Intermolt (C) 2-3 9 2 4
functional knowledge of molt staging
Early premolt (Do-D,) 4 9 3 4 whereby one can determine the stages (as
Late premolt (Dz-D~) 4 9 3-4 4 depicted in the present study) and their du-
Ecdysis (E) < I min 2 < I min I rations has many applications. Stage deter-
Cycle duration 13-14 21 11-12 5 minations of penaeid broodstock could be
a Except for ecdysis which is in minutes. Days are used for predicting larval production cycles,
rounded to the nearest integers. since open thelycum species do not mate
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MOLT STAGING OF P. SETIFERL'S A N D P. STYLIROSTRIS 183
STAQE C STAQE &Dj
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STMiE z
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44
FIGURE
1. Continued. zyxwvutsr
and closed thelycum species do not spawn observed in ponds as well (Shigueno 1975;
during late premolt or early postmolt. Since Apud et al. 1983). Additionally, because the
eyestalk enucleation or ablation during late market value is lower for shrimp harvested
premolt causes mortality, and during late with soft exoskeletons (Apud et al. 1983;
premolt causes delayed maturation, molt Baticados et al. 1986), it is important that
stage considerations prior to these proce- stage of molt be considered prior to harvest.
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dures may reduce losses and increase pro- Lastly, because crustaceans change physi-
ductivity. (However, it is probably not nec- ologically throughout the molt cycle, ex-
essary to microscopically molt stage an early perimental results (e.g., toxicity tests) may
postmolt shrimp since during this stage the
shrimp is soft, and the feel of it would suf-
fice.) Molt stages should be considered prior
to any stressful procedures such as shipping,
handling, therapeutic treatments or live-
harvesting. In ponds, determinations of molt
stages could aid in understanding molt syn-
chrony of pond animals and the possible
correlation of such cycling with lunar pe-
be affected by molt stage.
same was true for unilaterally ablated P.
semisufcatus (Browdy and Samocha 1985)
and P. monodon (Emmerson 1983). The
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In the present study, unilateral eyestalk
enucleation shortened the molt cycle of P.
setiferus by approximately two days. The
molt cycle durations of P. setiyerus and P.
stylirostris adults held at 27-29 C were 13-
riodicity. Boddeke et al. (1978) have dem- 14 days and 1 1-1 2 days, respectively (Table
onstrated molt synchrony in wild penaeid 1). Molt cycle durations have been deter-
populations, and molt synchrony has been mined for several penaeid species although
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184
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ROBERTSON ET AL.
comparisons are difficult since growth rate
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is size and temperature dependent. Juvenile
P. stylirostris (2.45 g) had a molt cycle which
lasted approximately nine days at 22.6 f 1
C (Huner and Colvin 1979) as compared to
11.5 days for our 52 g animals at 27-29 C.
Molt stage determinations can be made
of Sea Grant, U.S. Department of Com-
merce under Grant #NA83AA-D-0006 1.
Roy Garrett of Garrett Technical Illus-
tration designed the molt stage figure. We
are grateful to George Chamberlain, Jay
Huner, Larry Keeley, Susan Rankin and
Granvil Treece for reviewing the manu-
on live shrimp to allow repetitive obser- script.
vations, and individual shrimp can be mon-
itored if they are isolated or tagged. Obser- Literature Cited
vations can be made by placing the shrimp
Anderson, S. L., W. H. Clark, Jr. and E. S. Chang.
z
in a simple restraining device (Olivier and 1985. Multiple spawning and molt synchrony in
Salinger 1985) or by wrapping them in a a free spawning shrimp (Sicyonia ingentis: Pe-
moist paper towel (Huner and Colvin 1979; naeoidea). Biological Bulletin l68:377-394.
Smith and Dall 1985). Alternatively, por- Apud, F., J. H. Primavera and P. L. Torres, Jr. 1983.
Farming of prawns and shrimps. Southeast Asian
tions of the uropods can be excised, placed
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Fisheries Development Center. Extension Manual
in seawater in petri dishes and observed
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( 5 ) : 1-67.
when convenient. Such samples can be held Aquacop. 1977. Observation sur la maturation et la
refrigerated for up to 24 h without affecting reproduction en captivite des crevettes penaeides
the stage determination. Special equipment en milieu tropical. Actes de Colloques du CNEXO
4: 157-1 78 (in French with English abstract).
needs for molt staging include only a mi- Aquacop. 1979. Penaeid reared broodstock: closing
croscope of appropriate magnification ( I 0- the cycle of Penaeus monodon, P. stylirostris and
100 x). The time involved in staging in- P. vannamei. Proceedings of the World Maricul-
cludes sample preparation and sample ob- ture Society 10:445-452.
servation, both of which can be accom- Baticados, M. C. L., R. M. Coloso and R. C. Duremdez.
1986. Studies on the chronic soft-shell syndrome
plished in minutes. A few studies report stage in the tiger prawn, Penaeus monodon Fabricius,
determination by observations of pleopods from brackishwater ponds. Aquaculture 56:27 1-
(Schafer 1968; Longmuir 1983), however, 285.
the lack of pigmentation in the pleopods of Boddeke, R., R. Dijkema and M. E. Siemelink. 1978.
P. setgerus and P. stylirostris prevented The patterned migration of shrimp populations: a
comparative study of Crangon crangon and Pe-
staging using the criteria presented in the naeus brasiliensis. F A 0 Fisheries Report (200):
present study. 3 1-49.
Molt staging can be practiced with little Browdy, C. L. and T. M. Samocha. 1985. The effect
investment in equipment or time and has of eyestalk ablation on spawning, molting and
many applications in aquaculture as a rou- mating of Penaeus semiculcatus De Haan. Aqua-
culture 49: 19-29.
tine procedure. Molt staging will certainly Crocos, P. J. and J. D. Kerr. 1983. Maturation and
serve as a valuable aid in broodstock and spawning of the banana prawn Penaeus merguien-
pond management. sis de Man (Crustacea: Penaeidae) in the Gulf of
Carpentaria, Australia. Journal of Experimental
Marine Biology and Ecology 69:37-59.
Acknowledgments Drach, P. 1939. Mue et cycle d’intermue chez Crus-
taces decapodes. Ann. Inst. Oceanogr. 19: 103-39 I .
This work was sponsored in part by grants Emmerson, W. D. 1980. Induced maturation of prawn
from the Caesar Kleberg Foundation for Penaeus indicus. Marine Ecology Progress Series
Wildlife Conservation to Texas A&M Uni- 2:121-131.
versity, Addison L. Lawrence, principal in- Emmerson, W. D. 1983. Maturation and growth of
ablated and unablated Penaeus monodon Fabri-
vestigator, the Cullen Foundation, and Tex- cius. Aquaculture 32:235-24 I .
as A&M University Sea Grant College Huner, J. V. and L. B. Colvin. 1979. Observations
Program, supported by the National Ocean- on the molt cycles oftwo species ofjuvenile shrimp,
ic and Atmospheric Administration, Office Penaeus californiensis and Penaeus stylirostris
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MOLT STAGING OF P. SETIFERUS A N D P. STYLIROSTRIS
(Decapoda: Crustacea). Proceedings of the Na-
tional Shellfisheries Association 69:77-84.
Longmuir, E. 1983. Setal development, moult-stag-
ing and ecdysis in the banana prawn Penaeus rner-
guiensis. Marine Biology 77: 183-190.
Olivier, L. S. and M. A. Salinger. 1985. A simple
of the molting cycle of Penaeus duorarum, by mi-
croscopic examination of the setae of the endopo-
dites of pleopods. F A 0 Fisheries Report 57:381-
391.
Shigueno, K. 1975. Shrimp culture in Japan. Asso-
ciation for International Technical Promotion,
185
restraining device for the study and manipulation Tokyo, Japan.
of live penaeid shrimp. Progressive Fish-Culturist Smith, D. M. and W. Dall. 1985. Moult staging the
46(3):205-206. tiger prawn Penaeus esculentus. Pages 85-93 in P.
Read, G. L. H. 1977. Aspects of lipid metabolism in C. Rothlisberg, B. J. Hill and D. J. Staples, editors.
Penaeus indicus Milne Edwards. Masters thesis. Second Australian National Prawn Seminar, NPSZ,
University of Natal, Natal, South Africa. Cleveland, Australia.
Schafer, H. J. 1968. The determination of some stages
