1 JUNE 1999
Date: 20 May 1999
From: "Perry, Lee" <PerryL@prose.dpi.qld.gov.au>
To: <CRUST-L@vims.edu>
QUESTION:
I am a PhD student at the University of Queensland, Australia, and I am conducting research into moulting problems observed in Moreton Bay bug phyllosoma (Thenus orientalis and T. indicus). In particular, I am investigating a moulting problem known as Moult Death syndrome (MDS), which, as the name suggests, results in the death of an animal during the moulting process. During MDS a larva is unable to extricate itself from the old exoskeleton. This mortality, which occurs, most often, during the moult to the 4th and final phyllosomal instar, can be dramatic, with 80-100% of larvae dying in mass rearing systems. I am attempting to provide insight into the cause of MDS so that the effects of this mortality can be reduced.
As my studies progress, I am becoming increasingly interested in how
hormones might be implicated in this moulting problem. My initial literature searches revealed that work has been conducted on determining the types of hormones and the titres of moulting hormone (Christiansen 1988). For example, researchers have shown that 20-hydroxyecdysone is the main ecdysteroid hormone, with ecdysteroid titres changing throughout the moult cycle of the spider crab. Minimal levels of ecdysteroid are observed during early postmoult and maximum levels are observed during premoult (Spindler and Anger 1986).
In terms of the hormonal changes in larvae suffering from moulting
problems, relevant literature I have found so far is limited to work completed by Christiansen et al. (1984). Christiansen et al. (1984) found that crab larvae which die during ecdysis, have normal secretions of moulting hormone, however, the animals died during ecdysis, only after having being exposed to the insecticide diflubenzuron.
I was just wondering whether anyone had any ideas about the changes in hormone levels in moult death syndrome larvae, or indeed, larvae, which suffer from any type of moulting difficulty. Also, would the
differences in hormone levels be found during the entire larval cycle, or more particularly during the moult? Information from research on any other relevant decapod crustacean larvae would be very welcome.
References
Christiansen, M. E. 1988. Hormonal processes in decapod crustacean
larvae. Symposium of the Zoological Society of London. 58. pp. 47-68.
Christiansen, M. E., Gosling, E. and Williams, M. A. 1984. Effect of
the insect growth regulator diflubenzuron (Dimilin(r)) on the uptake of
glucose and N-acetylglucosamine into the cuticle of crab larvae. Marine Biology. 83. pp 225-230.
Spindler, K D. and Anger, K. 1986. Ecdysteroid levels during the larval development of the spider crab Hyas araneus. Gen. Comp. Endocr. 64. pp. 122-128.
Lee Perry :
Department of Zoology
Queensland University
Brisbane, 4072, Qld
lperry@zoology.uq.edu.au
or
Bribie Island Aquaculture Research Centre
P.O. Box 2066
Bribie lsland, Woorim
4507 Qld
perryl@dpi.qld.gov.au
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COMMENTS 1:
This is to tell you that we recently worked on moulting problems in
lobster and that beside the usual hormonal question we found that the blood oxygenation status, just prior to ecdysis, is likely related to an
efficient moult. There is in fact a deep transitory hypoxia that is
caused by an enhanced diffusion barrier for oxygen at the gill level as well as a deterioration in pumping efficiency of the scaphognathites. This transient hypoxia directly acts, via the CNS, on the pyloric chamber activity which also, as you know, undergoes cuticle replacement during ecdysis. The consequence is that the pyloric chamber exhibits a special motor pattern at that time. We suggested that its role could be:
1-to compress the old pyloric cuticle into the lumen of the pyloric
chamber and thereby facilitate its eventual extrusion and
2-to sever contact with the more posterior non-moulting midgut.
I do not know what are the conditions with your larvae but may be it
could help. Here are the references:
Clemens, S., Massabuau, J.-C., Meyrand, P., Simmers, J. (1999)
Changes in motor network expression related to moulting behaviour in lobster : role of moult induced deep hypoxia. J. Exp. Biol. 202 : 817-827.
Massabuau Jean-Charles
<massabuau@lnpc.u-bordeaux.fr>
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COMMENTS 2:
I just thought that I would mention a couple of things about MDS in our larvae that might be interesting to you. Our larvae are very lethargic at the time of moult, and instead of kicking quickly out of their shell, they just kind of lie in the water column, and do not seem
in a rush to get out of their old shell, and they change shape as if they
are going to moult slowly, but eventually they just stop in mid moult and die. The larvae do not moult until later in the morning, and it almost appears as if they can not absorb water as well as usual, as sometimes the cephalic shield does not expand at the same rate all the way around and some parts of the shield expand faster than the rest, and you end up with larvae that have a cephalic shield that looks more like a flower with petals.
Lee Perry
lperry@zoology.uq.edu.au
or
perryl@dpi.qld.gov.au
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COMMENTS 3:
Obviously your larvae are already quite tired when they start to moult!
Oxygen is certainly one more additional problem among a lot of problems...
Massabuau Jean-Charles
<massabuau@lnpc.u-bordeaux.fr>
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COMMENTS 4:
Yes, it does seem as if the larvae are tired, even though up to this point they seem absolutely normal, and have been eating very very well. Comments on the list and discussions with researchers here at the centre, have encouraged me to look into the possibility that the oxygen level has, for some reason (?), become unusually limiting at this very stressful time (compounding the problem of oxygen limitation that occurs because of physiological changes at the moult).
Lee Perry
lperry@zoology.uq.edu.au
or
perryl@dpi.qld.gov.au
***************
COMMENTS 5:
Just to add to the discussion on MDS. I had some pre-adult mud crabs
(Scylla serrata) that I had obtained from an aquaculture centre for my
research into shell disease in mudcrabs. Over half of these crabs had shell lesions on their carapaces. Over the 3 months that they were kept they were fed a diet of prawns, squid and white pilchards daily.
However over half of these crabs suffered from MDS.
The crabs appeared active and ate readily prior to the molt, but failed to completely remove their exuviae. The deaths appeared to be indiscriminant of the presence of shell lesions.
There is a link with Dimilin not only causing MDS but causing shell
lesions. However I couldn't establish any exposure here. I was also under the impression that the half life of Dimilin (diflubenzuron) was very short.
Eric mentioned improving the diet. I wondered how long the crab would need to be on an improved diet to prevent the MDS. The deaths occurred up to 6 weeks after being introduced to this diet. I am feeding this same diet to a batch of juveniles with no problems so far.
Can anyone offer any insight into what may have happened?
Leonie Andersen
Mud crab Shell Necrosis
Centre for Environmental Management
Central QLD University
P.O. Box 1319
GLADSTONE QLD 4680
AUSTRALIA
Tel: (07) 49 707315
Fax: (07) 49 707207
<andersel@quoin.cqu.edu.au>
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COMMENTS 6:
I have had a similar problem with the larvae of several crab species. Development seems to proceed very well up until the megalopal moult. Larval survival and development rates are comparable and synchronised between various broods, and the larvae appear to have large lipid droplets (I presume they are lipids) below the carapace. However at the megalopal moult the larvae are unable to extricate themselves from the moult.
On a previous occasion I asked members of the group what the problem could be, and the general response was that it was a nutritional problem. Although this may be a problem, I don't suspect it as being the main problem. Several of the crabs that I have worked on have larvae that are easily able to feed on Artemia (previously I only fed with Artemia but am now using mixed Artemia and rotifer diets). Also I have successfully reared Upogebiid larvae on the same Artemia diet and in the exact same rearing conditions, and they do fine.
If this problem I am having is related to the MDS, my question is what
can I do about it??? Are there any suggestions?? I am getting rather
frustrated as I spend a huge amount of time rearing the larvae only for
this to happen, and feel I am wasting my time now.
Brent Newman
University of Zululand
South Africa
<bnewman@pan.uzulu.ac.za>
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COMMENTS 7:
Recent discussion on the list about MDS reminded me some work I did many years ago on mud crab Scylla larvae in China and I think I should add those information to the discussion. No doubt MDS is a very complicated and thus interesting phenomenon, it seems to me MDS often occurs at high rate during the later larval stage, and it thus presents a big trouble both in lab and hatchery - It is a real pain that all the efforts and investments are wasted at the last minute.
In mud crab Scylla, MDS is particularly a problem during larval metamorphic moulting from zoea to megalopa stage. As Jean-Charles has rightly pointed out, hypoxia probably is a very important cause. I remembered that in my temperature experiment, one of the trials was to put last zoeal stage larvae, mass reared at 27 C (optimal temperature), to varied temperature conditions and to observe the rate of success of metamorphosis. MDS seems to occur mainly at high temperature conditions, with none of larvae moulted successfully at 35 C and high mortality at 30 C (the larvae were maintained in fingle bowl and no aeration was provided). Most of the larvae died of MDS.
However, if put metamorphosed megalopae to 35 C, they survived very well. In contrast, at low temperature (15 C) all zoea successfully moulted to megalopae but metamorphosed megalopae suffered high mortality. I also experienced a similar situation in large-scale larval culture - it was a short accidental power cut at midnight when most larvae were to moult to megalopae and you can image what happened the next day. Though hypoxia is likely to be an important cause for MDS, apparently it is not the only cause. My impression is that MDS also occurred at other adverse conditions, such as when larvae were fed with a low nutritional diet (eg rotifers at later larval stage), but those seems not as dramatic as the high temperature effects. Anyway, these are only observations; I agree that for the ultimate cause of MDS, one may have to dig into hormone regulation for the answer.
Chaoshu Zeng
Seikai National Fisheries Research Institute
49 Kokubu-machi, Nagasaki-shi
Nagasaki 850-0951
JAPAN
Tel: +81-95-821-4494, +81-95-824-9335 (H)
Fax: +81-95-833-2695
<cszeng@snf.affrc.go.jp> or
<chaoshuzeng@hotmail.com>
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COMMENTS 8:
I have been working on the larval culture of the mud (mangrove) crab
Scylla serrata for some time and MDS has figured prominently in our research. And as already mentioned in the Crust-L exchanges, MDS was particularly a problem at the first metamorphosis moult, from Z5 to megalopa. Our work has determined that its occurrence is easily controlled through nutrition, without varying any other factors. Careful selection of the type (strain) of Artemia used stops MDS occurring. Manipulation of nutrition means the difference between 100% MDS and 0%. Having said this I still believe that MDS is a general symptom (like a fever in humans) that is a response to various stressors. In our case it was only nutritional stress affecting the
larvae.
Most people keep their larval cultures close to the saturation level of DO. I fail to see why water DO level would suddenly lower at the onset of moulting so that if the moulting larvae go through a transient phase of dangerously low oxygen exchange during moulting surely it is a disruption to the normal moulting process that has lead to this problem. The low DO is a symptom not the problem.
David Mann
Bribie Island Aquaculture Research Centre
PO Box 2066
Bribie Island Qld 4507
Tel: (07) 3400 2023
Fax: (07) 3408 3535
<MannD@prose.dpi.qld.gov.au>
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COMMENTS 9:
In the discussion on MPS, nobody has mentioned the issue of energetic as yet.
But first and to get back to the issue discussed by David (the low DO is a symptom, not a problem) I would add that low DO is both a problem and a symptom. Firstly it is a problem linked to temperature because even if one would make sure DO is at a saturation level in the culture vessel, the volume of DO in warm water is going to be less than in colder water which could be one of the reasons why MDS is commonly observed in crustacean reared at temperatures higher than optimal (this problem would also be enhanced in static systems without aeration). Secondly, the level of DO might be a symptom especially in static culture without aeration. In this sort of conditions, the level of DO is highly dependent on consumption by the animal itself. If the animal is placed in a stressful environment (inappropriate
light, temperature or water movement, starvation...) it will require and consume more oxygen (higher metabolic rate, excessive swimming activity) in search of better environmental conditions or feed. Therefore, if the animal contributes to lowering the DO level excessively because of its response to a stressful environment, David is correct and low DO becomes a symptom of MDS. But this was not the point I wanted to get to. As most of you mentioned, DO, temperature, nutrition or basically all environmental factors that
constitute culture conditions will result in MDS when they fail to meet the requirements of the larvae. One of the reasons might be that any of those factors will directly or indirectly upset the energetic balance (energy input (feed) = energy for vital functions (maintenance, swimming, assimilation of feed) + energy for growth). It is easy to imagine how a diet can directly affect this balance (low or high energetic content). However, pinpointing how temperature might affect the balance can be more difficult. Temperature can directly
affect energy expenditure via the metabolic rate and indirectly via its
effect on feeding, swimming activity or DO.
Once the balance is upset the animal will be in an awkward position when facing moulting, a process that is likely to require high amount of energy during a period when feeding is interrupted. When moulting is successful, an exhausted larva might not be able to resume feeding and will die of starvation. The question rising from this approach is why would larvae hormonally initiate moulting under such unfavorable conditions? We might then be getting to adaptation (or adaptability) and selection issues.
Michel Bermudes
Tasmanian Aquaculture and Fisheries Institute
University of Tasmania
Nubeena Crescent
Taroona, TAS 7053
Tel: : 03 62 277 263
Fax : 03 62 278 035
<Michel.Bermudes@utas.edu.au>
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COMMENTS 10:
I agreed with Michel that at the present time we know very few about MDS, probably it is difficult to conclude what is the symptom and what is the problem. But one thing seems certain, as David also pointed out, MDS is a general overt phenomenon that could have various causes. It is also clear that when environmental conditions are controlled at optimal, MDS can be avoided. In the temperature experiment I mentioned in my last mail, at optimal temperature range and even at 15 C which is unlikely to be optimal for mud crab larvae, here hardly any MDS occurred (at 15 C moulting rate was 100%!). As to why MDS occurred at high rates at high temperature, I feel that larvae required higher DO level during metamorphic moulting, is a more important reason than the DO was depleted by larval activity beforehand. Why I said this is because under same conditions, megalopae, which appear to have higher metabolic and activity level than zoeae, did survive well at 35 C. Anyway, it seems to me that various stressors have somehow disturbed the normal moulting process (possibly a hormone regulating pathway) which results in MDS, and my point is that manipulating temperature and oxygen level would provide a feasible and relative easy way to look into how the process is affected.
As to the issue brought up by Michel about timing of moulting in crustaceans, I would also like to have a few words on it. Although there are many reports on capability of Decapoda larvae to shorten or lengthen their intermoult duration in response to various cues, in other cases, endogenous timing appears to have upper hand, in other words, moulting is set to occur at a certain time by inherited biological clocks regardless outside conditions, and we have evidence on that (MEPS 136: 69-79; Mar. Biol. 128: 299-305, we have another paper under revision about that).
Finally, I would like to touch off another question: why certain larval stages seem to be more susceptible to MDS? Since MDS normally occurs more prominently at later larval stages, it suggests some sort of cumulative effect?
Chaoshu Zeng
<cszeng@snf.affrc.go.jp> or
<chaoshuzeng@hotmail.com>
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COMMENTS 11:
I have been following the discussion about your MDS problem in phyllosoma larvae. Of particular interest was your description of the aspect of the dying larvae taking this shape that you describe as "flower with petals". From what we have seen while working on larvae of different species, two hypotheses come to mind:
Either the larvae were dead before taking this shape (from other contributing causes, like hypoxia as proposed by Jean-Charles Massabuau) which originated from water exchanges in dead tissues encased in the half-rejected exuvia; another cause for death might be the presence of pollutants (pesticides, insecticides, metals,...) in the water.
Or the shape change was caused by an osmotic shock, such as a decrease in salinity, leading to water invasion in these (most probably) osmoconforming larvae, resulting itself in an inflated body jammed in the carapace and finally in the death of the larvae. Did you monitor the water salinity?
Two reviews which might be of interest to you:
Charmantier G. (1998). Ontogeny of osmoregulation in crustaceans: a review. Inv. Reprod. Dev., 33: 177-190.
Charmantier G., Charmantier-Daures M. (1998). Endocrine and neuroendocrine regulations in embryos and larvae of crustaceans. IRD, 33: 273-287.
Guy Charmantier
Lab. D'Ecophysiology des Invertebres
Universite de Montpellier II
CP 092, Pl. Eugene Bataillon
34095 Montpellier Cedex 5
France
Tel: 334 6714 3672
Fax: 334 6714 3031
<charmantier@univ-montp2.fr>
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COMMENTS 12:
As for the Artemia type that were able to halt the occurrence of MDS in crab larvae, we trialed different brands of commercially available cysts, most representing different geographic strains. Only 2 of these gave acceptable results and did not include either San Francisco Bay, and Great Salt Lakes types (mentioned by Brent). Of the 2 types that worked, the ironic thing is that both sources are no longer productive. One was from China and the other source was not disclosed by the company but I am reliably informed that production has ceased (environmental changes, pollution?). I couldn't believe that upon solving the problem, the gate was closed.
As for the difference between the types, we are currently on the trail.
The best guess is something to do with lipids but we are still to complete the biochemical side of the investigation. Of special note is that the level of critical fatty acids EPA and DHA (popular among fish heads) appear to have little to do with it. I hope to publish this work in the not-too-distant future.
At this stage I can only suggest trying a supplementary source of nutrition for the xanthid larvae, e.g. larval stages of marine molluscs, copepods, particulate diets.
Mann David
<MannD@prose.dpi.qld.gov.au>