Superpotent synthetic tripeptide mimics of the mud-crab pumping pheromone.

Soluble pheromones released by the mud crab, Rhithropanopeus harrisii (Gould) during egg hatching cause the female crab to contract her abdomen rapidly (the pumping response). This stereotypical behavior can be induced in the laboratory by exposing egg-bearing females to solutions containing certain amino acids or peptides. Twelve amino acids exhibited response thresholds of 10(-3)-10(-10) M, the most potent (norleucine, methionine and tryptophan) having a hydrophobic sidechain. Four synthetic tripeptides of the form Gly-X-Arg, where the hydrophobic residue X was isoleucine, norleucine, methionine or phenylalanine, were superpotent agonists over a limited concentration range. Gly-Ile-Arg was a significant agonist at 10(-17) and 10(-16) M, both Gly-Nle-Arg and Gly-Met-Arg at 10(-20) and 10(-19) M, and Gly-Phe-Arg at 10(-21), 10(-20) and 10(-19) M. At the subattomolar concentration of 10(-20) M the superpotent pheromone mimics Gly-Met-Arg and Gly-Phe-Arg produced not only a statistically significant increase in the relative number of pumping mud crabs but also a substantial increase in the pumping rate ratio. In contrast, at 10(-13) M the tripeptide Gly-Met(O)-Arg with an internal residue of methionine sulfoxide blocked the abdominal pumping response of egg-bearing mud crabs.

Chemical Mediation of Larval Release Behaviors in the Crab Neopanope sayi.

Control of egg hatching was investigated in ovigerous females of the crab Neopanope sayi. Larval release is a brief event, generally lasting less than 15 min, during which females perform stereotypic behaviors involving vigorous abdomen pumping. Substances released by hatching eggs (pumping factors) of N. sayi, Rhithropanopeus harrisii, and Uca pugilator, but not Sesarma cinereum, evoked these stereotypic behaviors (pumping response) in ovigerous N. sayi. Spontaneous pumping and responsiveness to pumping factors varied with the age of the embryos. These results indicate that the eggs release pheromones around the time of hatching, which supports the general model for egg-hatching control described for R. harrisii (Forward and Lohmann, 1983). The chemistry of N. sayi pumping factors was investigated, and the pumping response was used as a bioassay in this study. Pumping factors adsorbed to Amberlite XAD-7 resin and could be eluted from it with methanol. Size fractionation by cascade pressure dialysis showed that the active molecules were <1000 daltons. Acid hydrolysis followed by reverse-phase HPLC amino acid analysis showed that the biologically active fraction contained peptides. Cysteine, glycine, methionine, and isoleucine were the four most common amino acids in these peptides. The responsiveness of N. sayi to hatch water from R. harrisii, the general similarity of adsorptive characteristics of hatch waters from the two species toward XAD-7 resin, and the amino acid compositional analysis suggest that the pumping factors from both species are similar. This supports the hypothesis that N. sayi pumping factors are also small peptides, as was suggested for those of R. harrisii (Rittschof et al., 1985, 1989).

Larval release in brachyuran crustaceans Functional similarity of peptide pheromone receptor and catalytic site of trypsin.

Studies of crab egg hatching and larval release behavior in the crab,Rhithropanopeus harrisii, generated a model describing the process. In the model, carboxyl terminal arginine peptides serve as pheromones that synchronize larval release. In response to the peptides, the female performs Stereotypic larval release behavior and casts larvae into the water column. The peptides originate from trypsin-like enzymatic activity as part of the egghatching process. Hatching can be simulated experimentally by incubating ovigerous crabs in either bovine or porcine trypsin. The female performs the larval release behavior. Eggs detach from the female, and immobile larvae hatch prematurely. Preincubation of trypsin with trypsin inhibitors eliminates these effects. Approximately nanomolar concentrations of five different polypeptide trypsin inhibitors evoke the female's larval release behavior. Because both peptides and trypsin inhibitors evoke larval release behavior and because trypsin inhibitors bind to both the peptide receptor and the enzyme with high affinity, the receptor binding site and trypsin catalytic site must be very similar. A relationship between the binding site of a peptide receptor and the catalytic site of trypsin is postulated. The difference may be substitution by a basic amino acid for the catalytic site serine. Molecular graphics modeling indicates that all necessary conditions for receptor binding can be met by substitution with lysine for the active site serine in the trypsin catalytic site. This substitution eliminates catalytic activity, maintains the binding affinity for trypsin inhibitors, and increases binding strength for peptides.

Behavioral Responses of Crustacean Larvae to Rates of Temperature Change.

The ontogeny of behavioral responses of larvae of the crabs Rhithropanopeus harrisii and Neopanope sayi to rates of change in temperature were analyzed using a video system. A temperature decrease evoked an ascent in both species. The threshold rates of decrease for Stages I and IV zoeae of R. harrisii, and Stage I zoeae of N. sayi, were 0.06, 0.1, and 0.09°C min-1, respectively. Stage IV zoeae of N. sayi were unresponsive to any rate of decrease. Larvae descended upon a temperature increase. For Stages I and IV zoeae of R. harrisii and Stage I of N. sayi the threshold rates of temperature increase were 0.07, 0.24, and 0.18°C min-1, respectively. Stage IV zoeae of N. sayi were again unresponsive. In general, there was an ontogenetic change in responsiveness as Stage IV zoeae of both species were less sensitive than Stage I zoeae. The average absolute amounts of temperature change needed to evoke a response was independent of the rate of change at rates above threshold and ranged from 0.29 to 0.49°C for both species. A consideration of larval sinking rates and ascent speeds, as well as normal environmental temperature gradients, shows that larvae of both species can respond to the rates and amounts of temperature change found in their environments. These responses constitute a negative feedback system that could be used to regulate depth relative to temperature.

Respiration and osmoregulation of the estuarine crab, Rhithropanopeus harrisii (Gould): effects of the herbicide, alachlor.

1. The effects of a sudden decrease in salinity and exposure to sublethal concentrations of the herbicide, alachlor, on osmoregulation and respiration of the crab, Rithropanopeus harrisii, were studied. 2. Crabs were hyperosmotic regulators at salinities below 24 ppt and became hypoosmotic at higher salinities. Upon a salinity decrease from 20 to 1 ppt, crabs adjusted their haemolymph osmolality to a stable hyperosmotic level in 8 hr. Alachlor concentrations to 50 ppm did not affect this adjustment. 3. A salinity decrease from 10 to 0 ppt elevated VO2 and the critical oxygen tension. This response was unaffected by alachlor concentrations as high as 25 ppm.

Tidal vertical migration: an endogenous rhythm in estuarine crab larvae.

Field-caught larvae of the estuarine crab Rhithropanopeus harrisii have a tidal rhythm of vertical migration when maintained in constant conditions. Laboratory-reared larvae do not show this rhythm. Endogenous tidal vertical migrations aid the retention of these planktonic larvae in estuaries near the parent populations.

Effects of neurochemicals upon a dinoflagellate photoresponse.

Photoresponsiveness by Gymnodinium splendens Lebour was monitored quantitatively by a microscope-television system. Exposure to the catecholamines DOPA and Dopamine caused a decrease in light sensitivity, while 0.01 mM norepinephine, or isoproterenol did not affect photoresponsiveness. Classical catecholamine blocking agents, dichloroisoproterenol, propranolol, and dibenzyline, and an inhibitor of DOPA synthesis, alpha-methyl-rho-tyrosine, caused an increase in sensitivity. In addition, acetylcholine and an inhibitor of acetylcholinesterase activity, eserine, caused an increase in sensitivity, while an inhibitor of acetylcholine action atropine, had the opposite effect. These experiments suggest that G. splendens may have an antagonistic catecholamine-cholinergic system which participates in regulating photosensitivity.

Phototaxis in a dinoflagellate: Action spectra as evidence for a two-pigment system.

Action spectra were determined in the UV region of the spectrum for the first phase of the phototactic response (stop response) and for the phytochrome pigment associated with this response in the dinoflagellate Gyrodinium dorsum Kofoid. Differences between these action spectra indicate the participation of two pigments in phototaxis. Following R (620 nm) irradiation of the phytochrome, the stop response maxima occur at 470 and 280-nm; after FR irradiation they shift to 490 and 300-310 nm. These maxima suggest that the photoreceptor pigment for phototaxis is a carotenoprotein. The action spectrum shift following the different phytochrome conversions may represent a trans to cis isomer change by the carotenoid. The absorption maximum of PR in the UV appears to be at 320 nm, which is consistent with the shift of the R absorption maximum to shorter wavelengths (620 nm) as compared to higher plants. The PFR absorption maximum appears as a broad band between 360 and 390 nm. Comparison of PR to PFR conversions by different intensities of 620-nm and 320-nm light indicates that at lower intensities the logarithm of the threshold for the stop response is inversely proportional to the logarithm of the intensity of the sensitizing light. The ratio of response activation by R and UV light is about 4:1.

Change in the photoresponse action spectrum of the dinoflagellate Gyrodinium dorsum Kofoid by red and far-red light.

Using cessation of movement (stop-response) as an index for light reception by the dinoflagellate Gyrodinium dorsum Kofoid, an association was shown between the blue-light action-spectrum maximum for this response and prior exposure of the organism to red and far-red light. When the light energy necessary to produce a positive stop-response (above 50%) was used as a criterion for the threshold intensity, the cells were most sensitive to light of 470 nm following an exposure to red (620 nm) light; after a far-red (700 nm) light exposure, the threshold was lowest for light of 490 nm. A second response criterion was the time in darkness until a positive response could no longer be initiated upon stimulation. The response persists longer for 490 nm than for 470 nm after both red and far-red light exposure. This result can theoretically be attributed to thermal reversion of the proposed phytochrome from the Pfr to the Pr form. A two-pigment system in which a phytochrome works in combination with a blue-absorbing pigment may be involved in the photoresponse.

The circadian rhythm of a behavioral photoresponse in the dinoflagellate Gyrodinium dorsum.

The circadian rhythm of the photoresponse to blue light in the dinoflagellate Gyrodinium dorsum Kofoid was investigated by the use of a closed circuit television system. The initial cessation of movement upon stimulation (stop-response) was used as the index of light reception. Under constant dark conditions cells grown on a 12L:12D regime show an endogenous circadian rhythm in their stop-response with maximum responsiveness occurring approximately one hour before the beginning of the expected light phase. This rhythmic response was only observed if the cells were irradiated with red light (620 nm) prior to stimulation with blue light. After preirradiation both far-red reversibility and the shift in the stop-response action spectrum from 470 nm to 490 nm could also be demonstrated. These findings may be related to the diurnal migration of marine dinoflagellates.