Limb Autotomy as an Investigatory Tool: Host Molt-Stage Affects the Success Rate of Infective Larvae of a Rhizocephalan Barnacle

D. M. Skinner was the first biologist to systematically investigatethe effect of multiple limb autotomy on ecdysis in decapod crustaceans.She proposed the existence of (1) limb autotomy factor anecdysis(LAF_an) which initiates precocious molting, and (2) limb autotomyfactor proecdysis (LAF^pro) which postpones proecdysis. Meantime to ecdysis did not differ significantly among groups ofsmall juvenile Callinectes sapidus with zero, two, four, andsix limbs removed. Variance was significantly less for the groupmissing six limbs; i.e., autotomy of six limbs synchronizedthe molt-cycle. These patterns were consistent with the hypothesesthat LAF^an exerts an additive effect, i.e., more anecdysialcrabs enter proecdysis as more limbs are autotomized and thata delay of proecdysis occurs above a threshold (>4) of autotomizedlimbs. Multiple limb autotomy provides investigators with easyaccess to crabs in metecdysial molt-stages which is useful instudying interactions between rhizocephalan barnacles and theirhosts. While 71% of metecdysial C. sapidus exposed to infectivelarvae of the sacculinid rhizocephalan, Loxothylacus texanus,developed the external stage of the parasite, no similarlysizedanecdysial crab was parasitized, suggesting that these crabsare not susceptible to infection during anecdysis. Size of C.sapidus at infection was inversely proportional to the numberof ecdyses between infection and emergence of the parasite,but not correlated to final host size. These data suggest thatthere is a minimum threshold for host size; smaller hosts undergomore ecdyses before attaining the threshold. Rhizocephalansare being considered as biological control agents and thereis a need to understand how they find and infect hosts. Becauselimb autotomy is such a useful research tool, D. M. Skinner'scontributions to our understanding of how limb autotomy influencesthe crustacean molt cycle will continue to pay significant dividendsin crustacean biology.     

The role of the Y-organ and cephalic gland in ecdysteroid production and the control of molting in the crayfish,Orconectes limosus

Summary The production of ecdysteroids (monitored by RIA) by Y-organs and cephalic glands in vitro was measured and hemolymph ecdysteroid levels were determined in the crayfish,Orconectes limosus, both after eyestalk ablation and as a function of time during natural premolt. Y-organ synthesis of ecdysteroid increased in parallel with a rise in hemolymph ecdysteroid concentrations under both conditions, peaking in substage D₂ of premolt. Y-organ ecdysteroid output after eyestalk ablation was 3–4 times higher. Thus, removal of the inhibiting system of the eyestalk effectively removes not only the principal control but also any modulation of ecdysteroid secretion by the Y-organs. Ecdysteroid levels remained low in Y-organ-ectomized crayfish, although premolt was initiated in some animals. The cephalic gland does not appear to contribute to the regulation of molting inOrconectes limosus. The Y-organs, on the other hand, are a principal source of ecdysteroids which regulate the major synthetic activities of premolt.     

Regulation of the Crustacean Mandibular Organ

The crustacean mandibular organ (MO) produces methyl farnesoate(MF), a juvenile hormone-related compound thought to have rolesin crustacean reproduction and development. Therefore, the controlof MF production by the MO has been of considerable interest.Current evidence indicates that the MO is negatively regulatedby peptides present in the eyestalk (MO inhibiting factor, MO-IH).Several eyestalk neuropeptides have been identified that inhibitMF synthesis by MO incubated in vitro. The amino acid sequencesof these MO-IH peptides are similar to peptides in the crustaceanhyperglycemic hormone (CHH) family of neuropeptides. In addition,there appears to be a compound in the eyestalk that lowers hemolymphlevels of MF in vivo but does not directly affect the MO invitro. The inhibition of MF synthesis by eyestalk peptides involvesthe inhibition of farnesoic acid O-methyl transferase, the lastenzyme in the MF biosynthetic pathway. The activity of thisenzyme is affected by cyclic nucleotides, suggesting that thesecompounds may be involved in the signal transduction pathwaymediating the effects of MO-IH.     

Interactions Between Limb Regeneration and Molting in Decapod Crustaceans

Molting and regeneration of lost appendages are tightly-coupled,hormonally-regulated processes in decapod crustaceans. Precociousmolts are induced by eyestalk ablation, which reduces circulatingmolt-inhibiting hormone (MIH) and results in an immediate risein hemolymph ecdysteroids. Precocious molts are also inducedby autotomy of 5–8 walking legs; adult land crabs (Gecarcinuslateralis) molt 6–8 wk after multiple leg autotomy (MLA).Autotomy of one or more of the 1° limb buds (LBs) that formafter MLA before a critical period interrupts proecdysis until2° LBs re-regenerate and grow to the approximate size ofthose lost. Based on these observations, Skinner proposed thatlimb buds produce two factors that control proecdysial events.Limb Autotomy Factor–Anecdysis (LAF_an), produced by 1°LBs when at least five legs are autotomized, stimulates anecdysialanimals to enter proecdysis. Limb Autotomy Factor–Proecdysis(LAF_pro), produced by 2° LBs in premolt animals when atleast one 1° LB is autotomized, inhibits proecdysial processes.Initial characterizations suggest that LAF_pro is a MIH-likepolypeptide that inhibits the synthesis and secretion of ecdysteroidby the Y-organs.     

The mysid Siriella armata,a biological model for the study of hormonal control of molt and reproduction in crustaceans: a review

Summary

The mysid Siriella armata provides a new biological model for investigations on the molting and reproductive physiology in crustaceans. The main endocrine centres (Y-organ, mandibular organ, androgenic gland, X-organ and sinus gland) have been described and are available for experimentation. Experimental cautery of Medulla Interna-Medulla Externa-X-organ-sinus gland complex (MI-ME-X-organ-SG) of the eyestalk inhibited molt and brood production demonstrating that the complex plays a role in regulation, at least via a positive action upon the circulating ecdysteroids. In the present paper, the results already obtained are reviewed and the perspectives offered by this biological model discussed in reference to other crustaceans.     

The Pericardial Sacs of Ocypode in Relation to the Conservation of Water, Molting, and Behavior

The surface area of pericardial sacs of the semiterrestrialcrab, Ocypode cordimana, is greater than that of pericardialsacs of the intertidal crabs, Ocypode macrocera and Ocypodeplatytarsis. Responses of the pericardial sacs with relationto conservation of water and molting vary with the species.In the semiterrestrial crab, Ocypode cordimana, the pericardialsacs store water during proecdysis which is used to stretchthe new cuticle after ecdysis. In contrast, the pericardialsacs of the intertidal crabs, Ocypode macrocera and Ocypodeplatytarsis, do not store water during proecdysis. But the sacsof these crabs swell for a brief period during ecdysis, apparentlyto accommodate excess fluid entering the body while the legsare being withdrawn from the old exoskeleton. Although the significanceremains unknown, deposits of uric acid are found in the pericardialsacs of Ocypode cordimana but not in the pericardial sacs ofOcypode macrocera and Ocypode platytarsis. Exposure of specimensof Ocypode cordimana to dry sand reduces the swelling of pericardialsacs, while proecdysial growth of the limbs remains unaffected.The pericardial sacs of Ocypode cordimana swell at a rapid rateif the crabs are maintained in darkness rather than in light.Implications of the present findings are discussed with relationto the habitat of the crabs.     

Conserved role of cyclic nucleotides in the regulation of ecdysteroidogenesis by the crustacean molting gland

Molting processes in crustaceans are regulated by ecdysteroids produced in the molting gland (Y-organ), and molting is indirectly controlled by circulating factors that inhibit the production of these polyhydroxylated steroids. Two of these regulatory factors are the neuropeptides molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). CHH appears to inhibit ecdysteroidogenesis in the Y-organ through the activation of a receptor guanylyl cyclase. The signaling pathway activated by MIH, however, remains a subject of controversy. It is clear that neuropeptides inhibit ecdysteroidogenesis by simultaneously suppressing ecdysteroid biosynthetic processes, protein synthesis, and uptake of high density lipoproteins. Data demonstrate that cAMP is the primary regulator of critical catabolic, anabolic, and transport processes, which ultimately support the capacity for ecdysteroid production by the Y-organ. While cAMP also regulates acute ecdysteroidogenesis to some extent, data indicate that cGMP is the primary signaling molecule responsible for acute inhibition by neuropeptides. It is clear that the regulatory roles filled by cAMP and cGMP are conserved among decapod crustaceans. It is unknown if these complementary second messengers are linked in a single signaling pathway or are components of independent pathways activated by different factors present in extracts of eyestalk ganglia.     

Yolk Synthesis in the Marine Shrimp, Penaeus vannamei

Eyestalk neuroendocrine factors control specific yolk proteinsynthesis in the ovaries of the shrimp, Penaeus vannamei. Abioassay was developed to measure specific yolk protein synthesisin vitro. The eyestalk neuroendocrine complex may also producea peptide capable of stimulation of yolk synthesis.     

Proteomics and signal transduction in the crustacean molting gland

Regulation of the molting cycle in decapod crustaceans involves2 endocrine organs: the X-organ/sinus gland (XO/SG) complexlocated in the eyestalk ganglia and the Y-organ (YO) locatedin the cephalothorax. Two neuropeptides [molt-inhibiting hormone(MIH) and crustacean hyperglycemic hormone (CHH)] are producedin the XO/SG complex and inhibit ecdysteroidogenesis in theYO. Thus, YO activation is induced by eyestalk ablation (ESA),which removes the primary source of MIH and CHH. Cyclic nucleotides(cAMP and cGMP) and nitric oxide (NO) appear to mediate neuropeptidesuppression of the YO. Proteomics was used to identify potentialcomponents of signal transduction pathways ("targeted" or cell-mapproteomics) as well as assess the magnitude of protein changesin response to activation ("global" or expression proteomics)in the tropical land crab, Gecarcinus lateralis. Total proteinsin YOs from intact and ES-ablated animals were separated bytwo-dimensional gel electrophoresis and expression profileswere assessed by image analysis and gene clustering software.ESA caused a >3-fold increase in the levels of 170 proteinsand >3-fold decrease in the levels of 89 proteins; a totalof 543 proteins were quantified in total YO extracts. ESA inducedsignificant changes in the levels of 3 groups of proteins elutingfrom a phosphoprotein column and detected with phosphoproteinstaining of two-dimensional gels;     

Trimeric G proteins in crustacean (Callinectes sapidus) Y-organs: occurrence and functional link to protein synthesis

Crustacean Y-organs produce ecdysteroid molting hormones. Regulation of ecdysteroidogenesis appears to be complex, involving regulatory ligands (including but not limited to molt-inhibiting hormone, an eyestalk neurohormone) and the capacity of the Y-organs to respond to those ligands. Available data indicate cell signaling pathways involving cAMP, cGMP, or both may be involved in regulation of Y-organ function. Trimeric G proteins link receptor occupancy to regulation of intracellular cAMP levels. In studies reported here, we have assessed the occurrence of G proteins in blue crab (Callinectes sapidus) Y-organs, and the link of G proteins to Y-organ function. Bacterial toxin-catalyzed ADP-ribosylation revealed a PTX-sensitive (alpha i-like) protein in Y-organ membranes, but failed to reveal a CTX-sensitive (alpha s-like) protein in Y-organ membranes. Western blotting with primary antibodies raised against conserved regions of mammalian G proteins detected an alpha i-immunoreactive protein (approximately 40 kDa) and two alpha s-immunoreactive proteins (approximately 50 and approximately 57 kDa) in Y-organ membrane preparations. Incubation of Y-organ membrane fractions with cholera toxin significantly suppressed incorporation of [35S]-methionine into TCA-precipitable Y-organ proteins, but had no detectable effect on ecdysteroidogenesis in short-term (6 h) incubations. The combined results indicate that C. sapidus Y-organs possess both Gi and Gs proteins, and that alpha s is functionally linked to regulation of glandular protein synthesis.     

Endocrine Regulation of Lipid Synthesis in Decapod Crustaceans

The legulation of lipid synthesis in the hepatopancreas of thecrab Pachygrapsus crassipes and the crayfish Procambarus sp.was investigated. Although deatalking induces an increase inthe rate of ¹⁴C-1-acetate incorporation into lipid, injectionsof crustecdysone into intermolt animals fail to elicit a similarresponse. In addition, the increased rate of lipid synthesisinduced by destalking is unaffected by removal of the Y-organ.It would appear then that the increase in lipid synthesis characteristicof early premolt is not controlled by either crustecdysone orthe Y-organ. It is suggested that formation of acetyl-CoA byany one of a number of possible mechanisms may bethe mannerin which the increase in fatty acid synthesis is effected duringpremolt.     

Proteins of the Crustacean Exoskeleton

We describe here some of the components of the exoskeleton ofthe decapod crustacean with emphasis on the constituent proteins,including both structural and enzymatic. All four layers, butparticularly the inner three, of the exoskeletons of four brachyuranscontain high concentrations of proteins 31 kDa; the innermostmembranous layer is especially rich in such proteins. A numberof crab exoskeletal proteins resemble insect cuticle proteinsin size (M_r) and isoelectric point (pI). A further similarityis the cross reactivity of crab exoskeletal proteins with fourdifferent antibodies against cuticular proteins of two speciesof insects. One of the small M_r exoskeletal proteins in theBermuda land crab Gecarcinus lateralis has a similar distributionas a protein of similar size in the cuticle of the tobacco hornworm Manduca sexta. The partial dissolution of an old exoskeletonand formation of the two outer layers of a new exoskeleton aremajor events in readying a crustacean for the increase in sizethat occurs at each molt. Expressing both parallel and sequentialactivation of a number of genes, a single layer of epidermalcells that bounds a crustacean such as G. lateralis synthesizesspecific proteins at different stages of the intermolt cycleas the outermost epicuticle and exocuticle are formed duringproecdysis and as the endocuticle and membranous layer are formedduring metecdysis. Finally, two sets of proteinases isolatedfrom integumentary tissues of land crabs degrade the same exoskeletalproteins in vitro as are degraded in vivo during proecdysis.     

Functional relations of crab molt-inhibiting hormone and neurohypophysial peptides

Biological and immunological relationships between molt-inhibiting hormone (MIH) activity in eyestalk ganglia extracts of the crab, Cancer antennarius Stimpson, and peptides of the vasopressin-oxytocin family were assessed. Lysine vasopressin (LVP), arginine vasopressin (AVP), vasotocin (VT), and oxytocin (OT) mimicked MIH action by inhibiting ecdysteroid production of Y-organ segments in vitro with the relative potencies LVP greater than AVP greater than VT much much greater than OT. The inhibitory effect was reversible and specific (6 other peptides did not alter Y-organ activity). MIH and LVP increased Y-organ cyclic adenosine 3',5' monophosphate (cAMP) levels dose-dependently and with identical time course in which the rise in cAMP preceded inhibition of ecdysteroid production. The synthetic vasopressin antidiuretic agonist 1-deamino-8-D-AVP (dDAVP) inhibited Y-organ steroidogenesis dose-dependently; the vasopressin analog ([1(B-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine[AVP) (d(CH2)5Tyr(Me)AVP), a vasopressor antagonist, had no effect on basal or MIH-suppressed steroidogenesis. AVP antiserum abolished the inhibitory action of MIH, LVP, and AVP. Competitive binding curves for MIH, LVP, AVP, VT, and OT with the AVP antiserum suggested that MIH is most closely related to LVP. MIH may be structurally related to the vasopressins and act on Y-organ cells via type V2 (cAMP-linked) receptors.     

Methyl farnesoate induced ovarian maturation in the spider crab,Libinia emarginata

Summary

To overcome the problem of getting crustaceans to reproduce in captivity, eyestalk ablation or X-organ sinus gland removal is commonly utilized in commercially important species such as shrimp. We have investigated the effect of unilateral and bilateral eyestalk ablation on methyl farnesoate (MF) production by mandibular organs (MOs) and on ovarian maturation in female spider crabs Libinia emarginata, a useful model since these animals are in a terminal molt and are devoid of a functional Y-organ. Non-reproductive, over-wintering female L. emarginata were induced to be reproductive by feeding and increasing the holding temperature to stimulate the endocrine system. In addition, we removed X-organ sinus glands by eyestalk ablation either unilaterally (UEA) or bilaterally (BEA) to further stimulate MF synthesis by MOs. Endogenous MF in the hemolymph was extracted and quantified by means of HPLC and in some cases by GC/MS. Oocyte growth and egg quality were studied simultaneously to determine how they were related to MF levels found during vitellogenesis. The initial MF concentration in unablated controls was low, 0.31 ng/ml of hemolymph, and this increased (p<0.05) to about 1 ng/ml by 2 weeks, remaining at about that level for the remainder of the experiment. Eyestalk ablation significantly stimulated MF concentrations by week 1 to nearly 2 and 3.5ng/ml in the UEA (p <0.01) and BEA (p <0.001) animals, respectively. Oocytes appeared to respond to increased MF levels, as ovarian maturation was initiated from the point at which MF increased (p <0.05). Thereafter, the rate of oocyte growth was directly correlated with the extent of elevation of MF. The gonado-somatic index [(GSI) = gonad weight/body weight × 100] of controls at the start was about 1.5 and increased to 6.5 by week 4. Mature oocytes were reached at a GSI around 7. Oocyte maturation was accomplished at week 2 in BEA, week 3 in UEA, and later than week 4 in controls. After maturation, oocytes started to degrade in some ablated animals, particularly in the bilaterally ablated ones where the highest MF concentrations were observed. These data indicate that MF elevations are required for stimulating ovarian maturation in Crustacea. MF appears to accelerate gonad development during the vitellogenic process, but may be deleterious at high concentrations. These results have a significant and important application and implications for aquaculture.     

Respiration and Mitochondrial Activity in Zea mays Roots As Affected by Osmotic Stress

Studies were made of metabolism in highly vacuolated and slightlyvacuolated Zea mays root tissue both during and after plasmolysis. Plasmolysis resulted in decreased respiration and carbon dioxideevolution from glucose and an increased sucrose synthesis. Inhibitionof respiration during plasmolysis in both the highly vacuolatedand slightly vacuolated tissue was not relieved by supply ofglucose, organic acids, or uncouplers of oxidative phosphorylation.Mitochondria isolated from plasmolysed tissue were tightly coupled,but activity in vitro was inhibited by exposure to a high negativeosmotic potential. It is suggested that low TCA cycle activityin vivo must be due either to inhibition of mitochondrial activityor to reduced flow of carbon through the glycolytic pathway. A low potential for TCA cycle activity after deplasmolysis issuggested, as addition of pyruvate stimulated carbon dioxideevolution but not oxygen uptake, which was severely decreased.This was presumably due to severe mitochondrial damage as shownby their activity in vitro. However, it is not clear whetherrespiration in vivo is rate limited by rapid leakage of metabolicintermediate (reported earlier) or by lysis of mitochondria. Deplasmolysis did not damage mitochondria from slightly vacuolatedtissue, a result which was consistent with respiratory measurementsmade in vivo. The data show that mitochondria in vacuolated tissue are damagedduring and after deplasmolysis and not before. It is suggestedthat lysis of mitochondria occurs in vivo as a result of a sharpincrease in the osmotic potential of the cell fluids.     

Uptake of high-density lipoprotein by Y-organs of the crab,Cancer antennarius. I. characterization in vitro and effects of stimulators and inhibitors

Cholesterol is the obligate precursor for ecdysteroid hormone synthesis by the ecdysial glands (Y-organs) in crustaceans, and all cholesterol in the hemolymph is bound to high-density lipoprotein (HDL). The mechanism was studied of how Y-organ cells acquire cholesterol. Y-organ segments were incubated with HDL isolated from hemolymph and labeled with ¹²⁵I. After incubation, tissue was homogenized in acid to determine radioactivity in acid-precipitable (cell associated, intact) HDL and in acid-soluble (degraded) HDL. Both HDL uptake and degradation showed saturation kinetics. At saturation most of the total counts represented degraded HDL; by 3 h, degradation was 80%. Rates of HDL uptake and breakdown were higher in Y-organs from de-eyestalked crabs (deprived thereby of molt-inhibiting hormone, MIH) than in glands from intact crabs. Both parameters were depressed by inhibitors of glycolysis and oxidative phosphorylation dose dependently and by low temperature. HDL uptake also was depressed by cAMP added to the medium experimentally or through efflux from the tissue during incubation. These results indicate a mechanism for HDL uptake that entails receptor-mediated, energy-dependent endocytosis of the entire HDL-cholesterol complex. Also the results suggest that HDL uptake and degradation are mediated by cAMP and depressed by an eyestalk factor, presumably MIH. © 1995 Wiley-Liss, Inc.     

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains

Development of a heterologous system for the production of homogeneoussugar structures has the potential to elucidate structure–functionrelationships of glycoproteins. In the current study, we usedan artificial O-glycosylation pathway to produce an O-fucosylatedepidermal growth factor (EGF) domain in Saccharomyces cerevisiae.The in vivo O-fucosylation system was constructed via expressionof genes that encode protein O-fucosyltransferase 1 and theEGF domain, along with genes whose protein products convertcytoplasmic GDP-mannose to GDP-fucose. This system allowed identificationof an endogenous ability of S. cerevisiae to transport GDP-fucose.Moreover, expression of EGF domain mutants in this system revealedthe different contribution of three disulfide bonds to in vivoO-fucosylation. In addition, lectin blotting revealed differencesin the ability of fucose-specific lectin to bind the O-fucosylatedstructure of EGF domains from human factors VII and IX. Furtherintroduction of the human fringe gene into yeast equipped withthe in vivo O-fucosylation system facilitated the addition ofN-acetylglucosamine to the EGF domain from factor IX but notfrom factor VII. The results suggest that engineering of anO-fucosylation system in yeast provides a powerful tool forproducing proteins with homogenous carbohydrate chains. Suchproteins can be used for the analysis of substrate specificityand the production of antibodies that recognize O-glycosylatedEGF domains.     

Small Heat Shock Proteins Protect Electron Transport in Chloroplasts and Mitochondria During Stress

Evidence indicates that small heat-shock proteins (Hsps) areinvolved in stress tolerance, but the specific cell componentsor functions that small Hsps protect or repair are mostly unidentified.We recently showed that the chloroplast small Hsps of higherplants (1) are produced in response to many environmental stresses(e.g., heat, oxidative, and high-light stress); and (2) protect(but do not repair) photosynthetic electron transport in vitroduring stress, specifically by interacting with the oxygen-evolving-complexproteins of Photosystem II (PSII) within the thylakoid lumen.However, in vivo evidence of the importance of these Hsps tophotosynthetic stress tolerance is lacking. Here we report positiverelationships between chloroplast small Hsp production and PSIIthermotolerance in (1) a heattolerant genotype of Agrostis palustris(bentgrass) and a heat sensitive genotype which lacks one ormore chloroplast small Hsps produced by the tolerant genotype;(2) ecotypes of Chenopodium album (lambs quarters) from thenorthern vs. southern U.S. (New York vs. Georgia); and (3) nineLycopersicon (tomato) cultivars/species differing in heat tolerance.These in vivo results are consistent with our previous in vitroobservations and indicate that genetic variation in productionof the chloroplast small Hsp is an important determinant ofphotosynthetic and, thereby, whole-plant thermotolerance. Recently,we showed that the mitochondrial small Hsp of plants protectsrespiratory (specifically Complex I) electron transport in vitroduring heat stress, and here we present evidence for previouslyunidentified small Hsps in mitochondria of mammal (rat) cellswhich also protect Complex I during heat stress. These resultssuggest that the mitochondrial small Hsps, like the small chloroplastHsps, are general stress proteins that contribute significantlyto cell and organismal stress tolerance.     

Hormonal regulatory role of eyestalk factors on growth of heart in mud crab,Scylla serrata

The present study was attempted to know the growth regulation of eyestalk factors on the growth of heart in Scylla serrata using eyestalk extractions and bilateral eyestalk ablations. The bilateral eyestalk ablation led to the maximum growth indices of the heart ((H) indices) to 0.162 and 0.158 in ablated male and female, respectively, in comparison to 0.153 and 0.167 in the control male and female and 0.147 and 0.157 in injected male and female, respectively. The data have shown that the heart of male crabs grows faster than female crabs. The study has also shown that bilateral eyestalk ablation resulted in a significant increase in the heart indices in males and has least effect on the growth of the female heart. The results presented strongly support a potential role of the eyestalk factors and molting hormone regulating the growth of the heart in S. serrata.