Hans Laufer

Identification of a juvenile hormone-like compound in a crustacean

Juvenile hormone (JH) has central roles in the regulation of insect development and reproduction but has not previously been identified in other arthropod classes. The hemolymph of a crustacean, Libinia emarginata (Leach), has now been analyzed for JH-like compounds. Samples contained 0.003 to 0.030 nanogram of JH III per milliliter and 10 to 50 nanograms of methyl farnesoate per milliliter; methyl farnesoate is a compound structurally related to JH III that has JH bioactivity. Several tissues were examined for synthesis and secretion of JH-like compounds. Of these tissues, only the mandibular organs produced and secreted JH III and methyl farnesoate. However, microchemical analysis revealed that this JH III was racemic, and thus likely an artifactual oxidation product of methyl farnesoate. Secretion of methyl farnesoate was related to reproduction in females, with the highest rates observed in Libinia near the end of the ovarian cycle when oocyte growth and vitellogenesis are greatest. These results indicate that JH-like compounds such as methyl farnesoate have regulatory roles in crustaceans.

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: The case of Bisphenol A

Background In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. Objectives We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. Discussion Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. Conclusions Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Methyl farnesoate and its role in crustacean reproduction and development

Studies with Libinia emarginata suggest that methyl farnesoate (MF), a product of the mandibular organs (MOs), may be a crustacean juvenile hormone. In order to better understand the significance of this compound in crustacean physiology, we first investigated the presence of MF in other decapods. MF was synthesized and secreted by MOs from all species tested. However, large differences in the level of MF secretion were observed between species and also between individuals of a species. For example, the level of secretion by MOs from L. emarginata was 100-fold greater than that observed in MOs from Homarus americanus. Analysis of hemolymph from these two species by GC-MS indicated comparable differences in the amount of MF present. Differences in the level of MF secretion by MOs from individuals of a species appear to reflect the physiological roles of this compound. For example, a close relationship was seen between MF secretion and gametogenesis in females of L. emarginata. Finally, treatment of lobster larvae with seawater containing MF caused a small but significant delay in their metamorphosis when compared with untreated larvae. These data suggest that MF affects reproduction in a manner similar to the effects of JH on insects, and may also have effects on the development of crustacean larvae. Taken together, these data support the classification of MF as a crustacean JH.

Polypeptides of fibroin and sericin secreted from the different sections of the silk gland in Bombyx mori

Abstract Secretory proteins, fibroin and sericin, extracted with disulfide cleavage from the lumen of several sections of the silk gland of the mature silkworm, Bombyx mori , were separated individually by gel electrophoresis at acid pH containing 4 M urea. Fibroin dissociated into two major polypeptides of light (f-2: 2.5·10 4 ) and heavy (f-1: 3.6·10 5 ) suggesting a multichain molecule cross-linked with disulfide bonds, while sericin is displayed as five polypeptides showing the molecular weight: 8.0·10 4 to 3.09·10 5 . The electrophoresis clearly demonstrated the secretion of different polypeptides from the section of silk gland; the posterior silk gland secretes two major (f-1 and f-2) and one minor (f-3) polypeptides of fibroin, the posterior section of the middle gland one polypeptide (s-4) of sericin, the middle section s-1 and s-3, and the anterior section s-2 and s-5, respectively. These polypeptides were characterized by the analyses of amino acid composition and the staining with PAS. Every five polypeptides, s-1, s-2, s-3, s-4 and s-5, secreted from the middle silk gland were similarly rich in serine, glycine and aspartic acid; and two polypeptides, s-1 and s-2, were stained in high intensity with PAS, and recognized as sericin. Two light polypeptides, f-2 and f-3, secreted from the posterior silk gland showed different composition from the reference data of fibroin and sericin, while the composition in the heaviest polypeptide, f-1, was identical with the reference data of fibroin.

Unifying Concepts Learned from Methyl Farnesoate for Invertebrate Reproduction and Post-Embryonic Development1

SYNOPSIS. Since the discovery that methyl farnesoate (MF), the unepoxidated form of the insect juvenile hormone (JHIII), is produced by mandibular organs of numerous crustaceans, extensive evidence has accumulated that this compound appears to perform similar functions in the Crustacea as JH performs in insects. A major function of MF appears to be in enhancing reproductive maturation. This was first shown by indirect experimentation with eyestalk ablation, which augmented MF production. Subsequently, direct treatments of several species of crustacea with MF showed that reproductive maturation was enhanced. A second function of MF, similar to that of the JH of insects, is in the maintenance of juvenile morphology. This is especially true in the late larval transformations into juveniles, where MF plays an inhibitory role, as well as during the transformation of juveniles into adults. These results were inferred from eyestalk removal experiments. In the case of the larval-juvenile transition, inhibitory results were also obtained with MF by direct hormone treatments. However, the transition from very early larval stages, such as one nauplius stage proceeding to the next, which in many cases also involves morphogenetic changes, may be occurring in the presence of MF. Indeed, MF appears to be stimulatory to early postembryonic larval stages of Crustacea. Again, this function of MF in Crustacea appears to be similar to functions of JH in early postembryonic insects. However, it should be pointed out that there are many more ‘‘early’’ stages in Crustacea than there are in insects, and very few of these cases have been investigated. When considering the animal kingdom and larval metamorphosis, the question may be raised whether there are other members of the JH family regulating metamorphosis and reproduction. One plausible example appears to be among certain annelids. The trochophores of Capitella respond to various juvenoids, but are most responsive, within one hour, to MF and eicosatrienoic acid. This latter compound is present also in adult annelids, where it has been named ‘‘Sperm Maturation Factor,’’ since it seems to function in the maturation of sperm in Arenicola. Therefore, eicosanoids perform in annelids two functions performed in insects by JHs. In conclusion, it seems that there are morphogenesis promoting responses to JHs in early larval development in crustaceans, annelids, and possibly other forms, which differ from those MF effects in later larvae of Crustacea where MF retards morphogenesis. Such early responses as noted here have recently also been described for insects. Furthermore, it is clear that the polyunsaturated 8,11,14-eicosatrienoic and aracidonic acids seem to be juvenoids, and appear to function as such in annelids, and may also be functionally active in insects and crustaceans. It seems reasonable to conclude therefore that new and novel juvenoids exist, while others still await discovery.

Vitellogenin synthesis by the fat body of the mosquito Aedes aegypti: Evidence for transcriptional control

Synthesis of vitellogenin (yolk protein) by the fat body of Aedes aegypti is triggered by the blood meal. Total RNA of the fat body begins to rise 2 hr post blood meal (PBM) and increases 3-fold by 12 hr. Vitellogenin synthesis is detectable 3–4 hr PBM, and reaches a peak by 28 hr PBM. After 28 hr PBM both total RNA and the ability to synthesize vitellogenins fall precipitously. Actinomycin D at 10 μg/ml inhibits RNA synthesis by about 90%, but does not inhibit in vitro synthesis of tissue proteins. At this concentration of actinomycin, vitellogenin synthesis remains constant in vitro for up to 6 hr, suggesting the presence of a relatively long-lived messenger RNA. When injected into mosquitoes, actinomycin prevents the normal increase in the rate of vitellogenin synthesis but allows synthesis to proceed at the rate occurring at the time of injection. The results suggest that the blood meal triggers the synthesis of both messenger and ribosomal RNA necessary for later vitellogenin synthesis.

Methyl farnesoate: Its site of synthesis and regulation of secretion in a juvenile crustacean

Abstract Analogy to Insecta suggests that JH(s) may play important roles in the development and reproduction of Crustacea. It has recently been shown by Laufer et al. (1987), using LC and GC/MS that the mandibular organ of adult crabs synthesized methyl farnesoate, the unepoxidated from of JH III, and that synthesis varied according to the sex, as a function of eyestalk ablation, and stage of vitellogenesis. In experiments reported here, we found that eyestalk removal from juvenile spider crabs, Libinia emarginata , resulted in a two-fold increase in the rate of MF synthesis by MOs in vitro . Furthermore, 2 h incubations of MOs from eyestalk ablated animals with eyestalk extracts inhibited the rate of synthesis of MF by about 60%. These results suggest that MF may be a gonad stimulating hormone (GSH) reported by other workers. Secretion by the MO is inhibited by an eyestalk factor (MO-IH), and MO-IH may be similar or identical to the gonad inhibiting hormone (GIH) reported by others.

Uptake of the yolk protein, lipovitellin, by developing crustacean oocytes☆

Abstract A variety of cytochemical techniques were used to demonstrate how crustacean lipovitellin accumulates within the egg. It was found that a protein serologically identical to the lipovitellin of yolk spheres was present in the hemolymph of vitellogenic crustaceans, but was absent from the hemolymph of males and immature females. In the three crustacean species studied ( Uca pugilator, Cambarus clarkii , and Libinia emarginata ), pinocytosis of fluorescein-conjugated lipovitellin and trypan blue occurred only during those periods when oocytes were accumulating yolk. It may be concluded from the present studies that yolk spheres develop in crustacean eggs primarily through micropinocytotic uptake of lipovitellin from the hemolymph, although other oocyte proteins appear to be made in the oocyte.

Isolation and characterization of sinus gland neuropeptides with both mandibular organ inhibiting and hyperglycemic effects from the spider crab Libinia emarginata

Using two-step reverse phase HPLC, we isolated and purified three peptides with mandibular organ inhibiting hormone (MO-IH) activity from the spider crab Libinia emarginata. One of the peptides, P22, gave a yield of 355 ng/SG. The others gave lower yields: P21, 9 ng/SG; P25, 67.5 ng/SG. The molecular weight was determined to be 8,439 for P25, 8,474 for P22, and 8,398 for P21 by mass spectrometry. All three peptides have similar amino acid compositions and contain 72–76 residues. We believe these peptides to be different isoforms of one family. The MO is more sensitive to the two minor isoforms, P21 and P25. All three isoforms can inhibit MO activity to a maximum inhibition of 70%. All three isoforms gave a significant hyperglycemic effect when injected into de-eyestalked fiddler crabs Uca pugilator. We believe the MO-IHs to be members of the crustacean hyperglycemic hormone (CHH) family, having similar amino acid compositions and both biological activities.

Effect of Methyl Farnesoate on Late Larval Development and Metamorphosis in the Prawn Macrobrachium rosenbergii (Decapoda, Palaemonidae): A Juvenoid-like Effect?

Methyl farnesoate (MF), the unepoxidated form of insect juvenile hormone III, was detected in larvae of the freshwater prawn Macrobrachium rosenbergii, which metamorphose to post-larvae following 11 larval stages. The possible role of MF as a morphogen was studied by administering the compound to M. rosenbergii larvae via an Artemia vector. Higher MF levels caused earlier retardation of late larval growth, and the highest dose retarded larval development. Furthermore, MF significantly affected the patterns of metamorphosis and the appearance of intermediate individuals exhibiting both larval and post-larval morphology and behavior. Three intermediate types were defined, two of which were found only at the MF-treated groups and one that was exclusive to the higher dose treatments. The relative abundance of intermediate specimens increased from 2% in the control to 32% in the high MF concentration, which suggests that MF has a juvenoid-like effect in this decapod crustacean.