Settlement cues are often the most difficult thing to provide to planktonic larvae, and are often a major hurdle to successfully rearing a new species of marine invertebrate in the lab. I have had several species that despite great success with the growth and survival of the larvae to competence, to my great frustration, I was never able to locate any substrate on which the larvae would complete metamorphosis. This is part of the challenge of rearing marine invertebrates!

A number of species are known to settle and metamorphose in response to red coralline algae. I tried live rock covered with red corraline algae with L. wurdemanni but the coralline algae did not appear to have any effect on settlement and metamorphosis.

Most of the species that have been demonstrated to respond to coralline algae either prey upon it directly or require a competition-free substrate on which to grow. Lysmata would not fit either category. Larval settlement inducers can be thought of as sign-posts that this location is going to be a good place to grow up. The presence of red coralline algae does not necessarily tell a shrimp larva anything about the suitability of the habitat for post-larval survival and growth. In general, the few inducers that have been identified are related to either the presence of a specific prey item, or the presence of others of the same species. The isolation and identification of settlement inducers is very difficult and hunting for an inducer for a species without obvious and specific settlement requirements has not even been attempted to the best of my knowledge.

The stuff in the coraline is related to GABA. GABA is a neurotransmitter used in commercial abalone production for larval settlement and metamorphosis.

Actually, I take issue specifically with this information. While it is true that the larvae of many coral and mollusc species do respond to the presence of coralline algae as a settlement cue, the implication of GABA in that behavior is still of great debate among larval biologists. There has never been GABA or any close GABA analog isolated from coralline algae, or (to the best of my knowledge) the surface of *any* marine organism. Given that, claims about the inductive ability of GABA as the "natural" inducer (or an analog) appear anecdotal at best.

In the case of sand castle worms (Phragmatopoma), for which a series of naturally occurring compounds (free-fatty acids) was subsequently isolated from the tubes of the adults and found to induce high proportions of settlement, it is doubtful that the compounds responsible for inducing settlement function in any way analogous to GABA. In fact, the settlement response of red abalone in the presence of GABA was duplicated by Chia & Koss by simply dropping the larvae in freshwater. Some researchers have interpreted this to be an indication of "stress" settlement along the lines of forced settlement by introduction of potassium-rich seawater. As people begin to research settlement cues and the role of bacteria, more and more studies are implicating the *bacteria* as an important component of natural settlement.

Even for the serpulid tubeworm, Hydroides dianthus, which is highly gregarious and for which an inductive cue has been extracted from the animals themselves, the presence of a microbial film is a *pre-requisite* for the response to the inductive cue (i.e., give the cue with clean glass slides and you get no settlement, take those clean slides out of the dish and add ones that have been coated with bacteria and the larvae settle immediately). For the congener, H. elegans, there is no specific gregarious response, but larvae respond specifically to several strains of bacteria isolated from the waters of Pearl Harbor.

The bottom line is that despite 20 years of study, we still know very little about the inductive cues and the neurological pathways associated with larval settlement, and some of the early information that we think we know may actually be misleading. There are only a *very* few cases in which a compound has actually been isolated from the substrate to which the larvae respond, and to the best of my knowledge there is only *ONE* identified chemical inducer that is not actively debated in the literature.....

I think the Waikiki aquarium has L. amboinensis settling in like 42 days. So maybe all of this is deranged and like the beer commercial, it's the water that counts.

That's what I'm told -- of course, they were using 1000G flow-through systems with natural seawater, and the larvae were fed heavily on Tetraselmis and rotifers, switching to enriched Artemia at around 6 weeks (time of settlement). Who knows what physical and/or chemical cues were present or pumped through the system? Fortunately, Lysmata is one of the species that spawn regularly within the tank, and we get many attempts at "getting it right."

Just my $0.02

Point taken. My thought was that the red coralline algae might indicate to the shrimp larvae that it was over a coral reef and I thought it was worth a try. I have also tried having an adult in the same water as the larvae and that did not appear to have any effect on settlement either. Do you have any ideas worth trying?

I apologize if that message came across as heavy-handed -- although it reads harshly, it is not how I intended it. I understand your logic here, and I wish that I had a better idea, but unless the larvae respond to water conditioned with the adults, the search for the settlement cue is likely to be microbial. If that is the case, it will be random luck whether or not you succeed in inducing high proportion settlement. The bright side is that even species with extremely specific settlement cues typically have some small percentage (~10% or so in the 1 species that has been specifically studied for this effect) of larvae that respond non-specifically to general microbial films. That means that the coralline algae substrate (which would have a bacterial film associated with it) should elicit some small amount of settlement from competent larvae. In experiments with these non-specific larvae, however, there was a relatively narrow window of time (~1wk) during which settlement in repose to bio-organic films took place, and after that time, the remainder of the larvae would die in culture if not provided with the appropriate cue for metamorphosis (in this case, living conspecific adults).

Re:GABA

Thanks for pointing this out. I'll leave the debate up to the larval biologists. I would just like to raise some shrimp and thought the coralline might work.

Sorry -- obviously a bit of a sore point with me. I've been involved in this debate for a little too long it seems, I apologize for that little diatribe.

That's why I made my comments about the water. Their water must have what ever the right stuff is. Unfortunately, my water does not have it. By the way, I was told they start giving enriched artemia at around 8 days

I'll get back to the water in my response to Jon below. As to the Artemia, I was told that they didn't start until about 6 weeks (which is on the order of the 42 days until they saw the first settlers). I'm not sure which is correct -- Maybe if you're following along on this thread, Bruce, you can check and confirm the technique for us? (I know he's really busy right now, so he may not be keeping up with list mail at all)

So the settlement cue is something that is in the water the entire larval time.

Who knows, it seems unlikely -- it's actually unlikely that some cue actually came in with the water in any case (there are only 3 cases known in which larvae respond to water soluble cues -- most require contact with the substrate). It's more likely that some organism (likely a bacterium) was brought in with the natural seawater and that was the cue to which the larvae respond.

I don't think anything changed in the water coming in from the outside which then triggered settling. How often do people see extended periods of the larval shrimp in suspension? I know in my experience with L.ambionensis, I had a dificult time keeping them alive more than 10-20 days, not waiting months hoping they would settle. Perhaps 40-60 days is about right for these shrimp.

It seems long for the planktonic larvae of a tropical species -- generally tropical species have shorter planktonic periods than their temperate cousins, and most temperate shrimps have 3 - 8 zoeal stages, and spend something on the order of 50-60 days in the water column before becoming competent to settle. Virtually every species with planktonic larvae studied to date have shown a dramatic ability to delay metamorphosis and prolong the pelagic period in the absence of appropriate settlement cues. In some species, the delay can extend the planktonic period by 10 times or more!

With some species, settlement increases with age, and the larvae become "less picky" but with most species assayed so far, if you don't give them the right cue, they keep waiting for it....