Cambarus Clarkii Research Articles

The features of an emerging whispovirus isolate from freshwater crayfish

A highly lethal freshwater crayfish Cambarus clarkii whispovirus (WSSV-Cc) was isolated from a naturally infected host. A large number of viral particles with typical nimavirus characters were observed in host cells. Genomic architecture and genetic evolution of this extremely virulent pathogen were uncovered. WSSV-Cc has a double-stranded DNA genome containing 287,179 bp, encoding 180 putative ORFs. Congruence analysis was performed between WSSV-Cc and known nimavirus genomes. The genome of WSSV-Cc is smaller than that found in isolates sampled before 2010, but is similar in size to that of recent isolates, which provided new evidence for the shrinkage of nimavirus genome size during evolution. Phylogenetic analysis with the complete genome also showed connections between genome size and evolutionary relationship. Twenty low-similarity genes and three variable regions were identified from the WSSV-Cc genome. In addition, two recombination events were detected in the WSSV-Cc genome, and analysis of the recombinant regions showed a close relationship among the three freshwater crayfish nimaviruses. These findings provide us the morphological, genomic, and genetic characters of an emerging whispovirus, which will broaden our understanding to nimaviruses and be useful for diagnosis of crayfish viral disease.

TAT-mediated oral subunit vaccine against white spot syndrome virus in crayfish

White spot syndrome virus is a highly pathogenic virus that infects crayfish and other crustaceans. VP28 is one of its major envelope proteins, and plays a crucial role in viral infection. Cell-penetrating peptides are short peptides that facilitate cellular uptake of various molecular cargoes, and one well known example is TAT peptide from HIV-1 TAT protein. In this study, recombinant plasmids were constructed and transformed into Escherichia coli strain BL21 (DE3) to express TAT-VP28, VP28, TAT-VP28-EGFP and VP28-EGFP fusion proteins. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry methods were used to confirm that TAT fusion proteins can translocate from the intestine to the hemolymph of the crayfish Cambarus clarkii. After immunization, activities of phenoloxidase and superoxide dismutase were analyzed, and it was found that rTAT-VP28 produced the most pronounced increase in both C. clarkii were vaccinated by oral administration of rTAT-VP28 and rVP28 for 7 and 14 days, and rTAT-VP28 resulted in the highest relative percent survival (RPS) (63.3% at 7 days, and 67.8% at 14 days), compared with rVP28 (44.4% at 7 days, and 53.6% at 14 days) following challenge with WSSV after the last day of feeding. This study reports the use of TAT-derived peptide as an oral delivery method of a subunit vaccine against WSSV in C. clarkii.

Surface-displayed VP28 on Bacillus subtilis spores induce protection against white spot syndrome virus in crayfish by oral administration

Surface-displayed heterologous antigens on Bacillus subtilis spores can induce the vertebrate animals tested to generate local and systematic immune response through oral immunization. Here, the protection potential of the recombinant spores displaying the VP28 protein of white spot syndrome virus (WSSV) was investigated in the invertebrate crayfish (Cambarus clarkii). The VP28 protein was successfully displayed on the surfaces of B. subtilis spores using CotB or CotC as a fusion partner. Crayfish were administrated orally by feeding the feed pellets coated with B. subtilis spores for 7 days and immediately followed by WSSV challenge. Oral administration of either spores expressing CotB-VP28 or CotC-VP28 resulted in significantly higher relative survival rates of 37.9 and 44.8% compared with the crayfish orally administrated with the spores nonexpressing VP28 (10.3% relative survival rate). When challenges were separately conducted at 7 and 21 days after oral administration, the relative survival rates increased to 46.4 and 50% at 7 days post-oral administration, but decreased to 30 and 33.3% at 21 days after oral administration. These evidences indicate that the surface-displayed VP28 on B. subtilis spore could induce protection of crayfish against WSSV via oral administration. This is the first report to use the spore surface display system to deliver orally a heterologous antigen in an aquatic invertebrate animal, crayfish. The results presented here suggest that the spore-displayed VP28 might be suitable for an oral booster vaccine on prevention of WSSV infection in shrimp farming.

Oral delivery of DNA vaccine encoding VP28 against white spot syndrome virus in crayfish by attenuated Salmonella typhimurium

Protective immune responses in shrimp induced by DNA vaccines against white spot syndrome virus (WSSV) with intramuscular injection have been reported in recent reports. In this study, we investigated the utilities of attenuated Salmonella enterica serovar Typhimurium (Salmonella typhimurium) as a bactofection vehicle for the oral delivery of a DNA vaccine plasmid to crayfish (Cambarus clarkii). The DNA vaccine plasmid pcDNA3.1-VP28, encoding viral envelope protein VP28, was transformed to an attenuated S. typhimurium strain SV4089 and the resulting recombinant bacteria named SV/pcDNA3.1-VP28 were used to orally immunize crayfish with coated feed. Successful delivery of the DNA vaccine plasmid was shown by the isolation of recombinant bacteria SV/pcDNA3.1-VP28 from the vaccinated crayfish. The distribution analysis of plasmid pcDNA3.1-VP28 in different tissues revealed the effective release of DNA vaccine plasmid into crayfish. RT-PCR and immunoflurescence results confirmed the expression of protein VP28 in the vaccinated crayfish. Challenge experiments with WSSV at 7, 15, 25 days post-vaccination demonstrated significant protection in immunized crayfish with relative survival rate 83.3%, 66.7% and 56.7%, respectively. Studies on stability and safety of SV/pcDNA3.1-VP28 showed the recombinant bacteria could exist in crayfish at least 7 days but not more than 10 days and without any observable harm to the host. Our study here demonstrates, for the first time, the ability of attenuated Salmonella as a live vector to orally deliver a DNA vaccine against WSSV into the arthropod crayfish and provides a new way to design more practical strategies for the control of WSSV and other invertebrate pathogens.

A new fluorescent quantitative PCR-based in vitro neutralization assay for white spot syndrome virus

A fluorescent quantitative PCR (FQ-PCR) assay utilizing SYBR green I dye is described for quantitation of white spot syndrome virus (WSSV) particles isolated from infected crayfish, Cambarus clarkii. For this assay, a primer set was designed which amplifies, with high efficiency and specificity, a 129 bp target sequence within ORF167 of the WSSV genome. Conveniently, WSSV particles can be added into the FQ-PCR assay with a simple and convenient method to release its DNA. To establish the basis for an in vitro neutralization test, primary cultures of shrimp cells were challenged with WSSV that had been incubated with a polyclonal anti-WSSV serum or with control proteins. The number of WSSV particles released from the cells after these treatments were assayed by FQ-PCR. This test may serve as a method to screen monoclonal antibody pools or recombinant antibody pools for neutralizing activity prior to in vivo animal experiments.

Transcriptional profile of shrimp white spot syndrome virus (WSSV) genes with DNA microarray

In an attempt to obtain the transcriptional profile of shrimp white spot syndrome virus (WSSV) genes, DNA microarray analysis was performed using amplified DNA fragments of the WSSV genome that covered most of the presumptive open reading frames (ORFs). Total RNAs were extracted from WSSV-infected crayfish (Cambarus clarkii) and reverse transcribed into cDNA and labeled with 32P-dATP. The DNA microarray detected 81.1% of the ORFs, and 47 of these were transcribed at 6 h post infection, suggesting they may be early genes. Some early genes were further confirmed by temporal analyses of gene transcription. Results in this study indicated the effectiveness of DNA microarray for discovering early viral genes.

Protection of crayfish, Cambarus clarkii, from white spot syndrome virus by polyclonal antibodies against a viral envelope fusion protein

White spot syndrome virus (WSSV) is a large double-stranded DNA virus, causing considerable mortality in penaeid shrimp and other crustaceans. WSSV produces five major structural proteins, including two major envelope proteins, VP28 and VP19. To produce VP28 and VP19 as a single protein for antibody production, DNA sequences encoding both open reading frames were fused together and cloned into pET-22b(+) expression vector. The fusion protein, VP(19+28), was expressed in Escherichia coli, purified using Ni2+ His affinity chromatography and injected into a rabbit. Antiserum collected from the immunized rabbit was tested in vivo for ability to protect crayfish, Cambarus clarkii, from disease caused by WSSV. Fifteen days after challenge with WSSV, treatment with VP(19+28) antiserum gave 100% protection against disease in the ambient temperature range of 15-22 degrees C and 65% protection at a constant temperature of 26 degrees C. These results demonstrated VP(19+28) antiserum is effective in protection of crayfish from WSSV and confirmed that VP19 and VP28 play an important role in WSSV host infection. Targeting both VP19 and VP28 may be effective for the design of both immunotherapeutic medicines and reagents to detect WSSV.

Purification and characterization of White Spot Syndrome Virus (WSSV) produced in an alternate host: crayfish, Cambarus clarkii

Penaeid shrimp is the natural host of White Spot Syndrome Virus (WSSV) that can cause high mortality in the infected hosts. Attempts to obtain sufficient amounts of purified intact WSSV for characterization have been unsuccessful. Using crayfish, Cambarus clarkii as a proliferation system, a large amount of infectious WSSV was reproduced and intact WSSV viral particles were purified with a new isolation medium by ultra-centrifugation. Purified WSSV particles were very sensitive to organic solvents and the detergent, Triton X-100. The size of the rod-shape, somewhat elliptical, intact WSSV was 110–130×260–350 nm with a long, tail-like envelope extension. The naked viral nucleocapsid was about 80×350 nm, and it possessed 15 spiral and cylindrical helices composed of 14 globular capsomers along its long axis, and a ‘ring’ structure at one terminus. Distinct WSSV genome DNA patterns were obtained when the purified genomic dsDNA of WSSV was digested with five different restriction enzymes ( HindIII, XhoI, BamHI, SalI, and SacI). In addition, at least 13 major and distinct protein bands could be observed when purified intact WSSV viruses were separated by SDS-PAGE followed by Coomassie Brilliant R-250 staining. The estimated molecular weights of these proteins were 190, 84, 75, 69, 68, 58, 52, 44, 28, 27.5, 23, 19, and 16 kD, respectively. Both the 44 and 190 kD proteins were easily removed if the hemolymph from the WSSV infected crayfish was transiently treated with 1% Triton X-100 before it was subjected to gradient centrifugation, indicating that both of them are located on the surface of the viral envelope. These characteristics are consistent with WSSV isolated from the penaeid shrimp.

Study of Joint Action of Mercury and Lead On Discharges of the Antennule-Inner Ramus Nerve of the Crayfish (Cambarus Clarkii)

Abstract The joint action of mercury and lead on the discharge of antennule-inner ramus nerve of crayfish (Cambarus clarkii) has been studied by means of a multichannel record of electrophysiological signals. the results show: 1. When the concentration of mercury (Hg2+) in water is only half of the restricted concentration (0.1 ug1-1) but there is also 12.5 ug1--1 lead (Pb2+) present, the joint inhibition of the two heavy metallic ions on the discharge is much stronger than that of the restricted concentration of mercury present. the slope of a linear regressive equation which indicates the frequency of discharges (Fn/F1) and action time is < −0.08129 ± 0.00357 ms. Further, in a certain concentration range, the higher the concentration of lead (Pb2+), the inhibition is stronger. 2. When the concentration of lead (Pb2+) in water is only 1/2 of the restricted level (100 ug 1-1Pb2+), but when there is 0.025 ug1-1 Hg2+ present, the joint inhibition of the two heavy metal ions on the discharge is much stronger...

Effects of a high molecular weight toxin from Physalia physalis on glutamate responses

The effects of a high molecular weight toxin from Physalia physalis (P 3) were investigated on glutamate evoked potentials in snail ( Zachrysia guanensis) neurons and in crayfish ( Cambarus clarkii) neuromuscular junction. The glutamate evoked potentials of snail neurons were reversibly blocked by P 3 in a dose-dependent manner (2–200 μM). A reversible blocking action was also found for P 3 on excitatory junctional potentials and on glutamate potentials of crayfish at a concentration range of 6 nM–60 μM. Experiments carried out with independent stimulation of the excitatory and inhibitory nerves showed that the effect of P 3 (60 nM–10 μM) was exerted predominantly on excitatory junctional potentials. However, at higher doses (> 10 μM) a slight reduction of the inhibitory potentials was also observed. These results suggest that P 3 reversibly blocks glutamate receptors. Thus, it could be a promising tool for further studies on glutamatergic transmission.

The uptake and release of glutamate at the crayfish neuromuscular junction.

The abdominal slow flexor muscle of the crayfish (Cambarus clarkii) was pre-incubated in deuterium-labelled glutamate (glutamate-d5) solution, and the release of glutamate, glutamate-d5 and aspartate in the bath solution was measured by mass fragmentography. The glutamate-d5 was taken up into the preparation and released together with endogenous glutamate. The resting release of glutamate-d5 was 8.2 +/- 0.96 pmol/10 min after the incubation with 0.5 mM-glutamate-d5, and those of endogenous glutamate and aspartate were 7.4 +/- 1.19 and 2.8 +/- 0.81 pmol/10 min, respectively. The release of glutamate-d5 was not significantly increased by nerve stimulation, while that of endogenous glutamate was increased by about 9.7 pmol/10 min above the resting release. The application of high-K solution induced a net increase of 1.9 pmol/10 min in glutamate-d5 release, 6.1 pmol/10 min in endogenous glutamate release and 7.9 pmol/10 min in aspartate release. The relative amount of glutamate-d5 accumulated into the preparation during pre-incubation with 0.5 mM-glutamate-d5 was a few per cent of the endogenous glutamate in the preparation. It is concluded that the exogenous glutamate is taken up mainly into a compartment which differs from that related to nerve evoked transmitter release and that high-K solution has effects on the amino acid release which differ from those of nerve stimulation.

On the quantal release of endogenous glutamate from the crayfish neuromuscular junction.

1. The abdominal slow flexor muscle was isolated from the crayfish (Cambarus clarkii) and placed in 150 microliters. Harreveld solution. The concentrations of glutamate and aspartate in this solution were measured by mass fragmentography. 2. Application of black widow spider venom (BWSV) produced a marked increase in the frequency of miniature excitatory post-synaptic potentials (m.e.p.s.p.s). During the high frequency discharge of m.e.p.s.p.s, the glutamate content in the solution was significantly increased. There was an approximately linear relationship between the increase in the glutamate efflux produced by BWSV and the variance of the membrane potential fluctuation during high frequency discharge of m.e.p.s.p.s. 3. In most cases, the efflux of aspartate during control rest periods was smaller than that of glutamate. During the discharge of m.e.p.s.p.s produced by BWSV, the increase in the aspartate efflux was very small compared to glutamate. 4. Nerve stimulation caused a significant increase in the efflux of glutamate, but the change in the aspartate efflux was very small and not significant. 5. Application of methylene blue increased the frequency of m.e.p.s.p.s and glutamate efflux, but little, if any, increase was found in aspartate efflux. 6. It is concluded that glutamate is preferentially released from nerve terminals in a quantal fashion.

Release of glutamate from the crayfish neuromuscular junction.

1. The superficial abdominal flow flexor muscle was isolated from the crayfish (Cambarus clarkii) and placed in a bath solution of 100 microliters. The concentration of glutamate in this solution was measured by mass fragmentography using a gas chromatograph-mass spectrometer. 2. The excitatory post-synaptic potential (e.p.s.p.) of the slow flexor muscle and its sensitivity to L-glutamate were similar to those observed in the opener muscle of the dactyl in the walking leg or claw of the crayfish. 3. The background efflux of glutamate during control rest periods was about 20 p-mole/10 min. Nerve stimulation caused a significant increase in the efflux of glutamate. The net release of glutamate above the background was 11.9 p-mole/100 microliters. at 10 Hz stimulation and 21.1 p-mole/100 microliters. at 20 Hz stimulation. 4. When the amplitude of e.p.s.p. was decreased by streptomycin, thereby reducing the muscle contraction, the net release of glutamate by nerve stimulation was not changed. Streptomycin depressed the e.p.s.p. by its action on the post-synaptic membrane. 5. When the external concentration of Ca was lowered, the amplitude of e.p.s.p. and the net release of glutamate were decreased. 6. It is concluded that L-glutamate is released from the nerve terminals of the crayfish neuromuscular junction.

Distribution and pharmacological properties of synaptic and extrasynaptic glutamate receptors on crayfish muscle.

1. The distribution of glutamate sensitivity was measured in the opener muscle in the walking legs of the crayfish (Cambarus clarkii). L-Glutamate was ionophoretically applied under visual control. 2. Bundles of a few muscle fibres were isolated and viewed with Nomarski optics. Two axons, presumably excitatory and inhibitory, branched widely over the surface of individual muscle fibres, forming numerous clusters of boutons or varicosities. 3. Glutamate sensitivity was measured from the slope of the dose-response curves obtained by ionophoretic application of L-glutamate and expressed as mV/nC. The highest sensitivities were about 100 mV/nC, obtained at the edge of synaptic boutons. The sensitivity declined to less than 5% about 2 micrometer away from the synaptic terminal. The time course of glutamate potentials was approximately the same as that of spontaneous synaptic potentials. 4. Glutamate depolarization started within 300 microsec after ionophoretic release of glutamate. This time lag was shorter than the synaptic delay of the nerve-evoked synaptic potential measured with an extracellular micro-electrode. This indicates that glutamate depolarization results from a direct action on the post-synaptic receptor. 5. Application of L-alpha-kainic acid decreased the amplitude of the glutamate potential produced at the synaptic region, whereas kainate increased the amplitude of the glutamate potential at the extrasynaptic region. It is suggested that the pharmacological properties of the extrasynaptic receptor differ from those of the synaptic receptor. Possible mechanisms for the different actions of kainate are discussed.

An analysis of the inhibitory post-synaptic current in the voltage-clamped crayfish muscle.

1. Inhibitory post-synaptic currents (i.p.c.s) were recorded from the feed-back current through a wire electrode inserted longitudinally into the opener muscle fibre of the claw in the crayfish (Cambarus clarkii). 2. I.p.s.c. rose to its peak in about 3-4 msec and decayed approximately exponentially. The decay time constant at -100 mV was 9.4 msec. 3. The decay time constant decreased as the membrane was hyperpolarized and increased during depolarization. The time constant (tau) depends on voltage (V) according to the relation tau = a exp (AV), with a = 18.6 msec and A = 0.0065 mV-1. Voltage dependence was opposite in direction to that seen at frog end-plates, but in the same direction as that of e.p.s.c. in crayfish muscle. 4. At lower temperatures, the rise and fall times of i.p.s.c.s were prolonged. Q10 for the decay time constant was 2.4 between 22.6 and 12.5 degrees C. 5. When pH was decreased from 7.2 to 5.5, the decay time constant increased by about 50%, with little change in the voltage dependence of the time course. 6. When chloride in the solution was changed to iodide, the decay time constant was increased by a factor of 3, while voltage dependence of the time course was not changed. In bromide solution the decay time constant increased by about 50%. 7. Peak amplitudes of i.p.s.c.s were approximately linear as the membrane was depolarized, but they levelled off as the membrane was hyperpolarized beyond reversal potential. The non-linear I-V relation did not result from inadequate voltage clamping, nor from a change in the inside concentration of chloride. After equilibration with iodide solution the I-V relation was approximately linear. 8. The decay time constant was increased after repetitive nerve stimulation. This prolongation became more pronounced at lower temperatures. 9. The kinetic process of the transmitter action is discussed. It is suggested that the rate limiting process for i.p.s.c. is binding and unbinding of the transmitter to the receptor.

Inhibitory postsynaptic current in voltage-clamped crayfish muscle.

THE current–voltage relationship provides important information about the mechanism of ionic permeation through membranes. In excitatory synapses the relationship between membrane potential and excitatory postsynaptic current (e.p.s.c.) has been extensively studied using the voltage-clamp method1–6. In the frog endplate it has been shown that the e.p.s.c. was smaller than would be expected from a linear relationship during hyperpolarisation of the membrane3,4. On the other hand, after iontophoretic application of acetylcholine, the synaptic current increased more than expected at hyperpolarised membrane potentials7. In investigating the current–voltage relationship it is important to measure the current on both sides of the equilibrium potential. Inhibitory postsynaptic current (i.p.s.c.) is more suitable for this purpose, because the reversal potential is near the resting potential and so the current–voltage relationship on both sides of the reversal potential can be measured using relatively small potential changes. Little attention has been paid, however, to inhibitory postsynaptic membranes, possibly because the inhibitory synapses are distributed diffusely over the surface of the cell or situated on dendrites remote from the cell body where the electrical changes are measured. In these conditions it is difficult to measure accurately the current–voltage relationship. We have cannulated the opener muscle of the claw in the crayfish (Cambarus clarkii) using a stainless steel wire, the membrane potential being clamped at various levels as described previously6. Although the inhibitory synapses are distributed over the whole surface of the muscle fibre, the space clamp condition of the muscle fibre was maintained well using this technique. We have found that the relationship between membrane potential and i.p.s.c. was highly nonlinear on hyperpolarisation of the membrane.

Permeability changes produced by L-glutamate at the excitatory post-synaptic membrane of the crayfish muscle.

1. Permeability changes produced by L-glutamate at the neuromuscular junction of the crayfish (Cambarus clarkii) were investigated by application of the drug iontophoretically to the voltage-clamped junction and measuring the resulting 'glutamate current'. 2. Reversal potentials were determined by measuring the glutamate current at different membrane potentials. They were +39-1 +/- 3-6 mV (mean +/- S.E. of mean) in normal solution and +16-5 +/- 2-0 mV in solutions made twice as hypertonic by the addition of sucrose. 3. Decreasing external Na+ concentration shifted the reversal potential in the negative direction; increased Na+ in the positive direction. 4. The relation between the amplitude of the glutamate current and extracellular Na+ concentration was approximately linear. 5. Alteration of the external K+ or Cl- concentration did not affect the amplitude or reversal potential of glutamate current. 6. In Na+-free solution the application of L-glutamate produced a small inward current at the resting potential and its amplitude was augmented by increasing the external Ca2+ concentration. 7. Increasing the Ca2+ concentration in the normal Na+ media produced no appreciable effect on the reversal potential but decreased the amplitude of glutamate current. 8. The results indicate that L-glutamate increases the membrane permeability mainly to Na+ and slightly to Ca2+. 9. The time course of glutamate current was shorter than that of the concentration calculated from the diffusion equation and it was simulated more closely by the square of the concentration.

Ionic mechanism of the excitatory synaptic membrane of the crayfish neuromuscular junction.

1. The reversal potential for the excitatory neuromuscular junction of the crayfish (Cambarus clarkii) was measured using the voltage clamp method. The potential change was recorded with an intracellular microcapillary and the negative phase of the output of the feed-back amplifier was connected to the stainless-steel wire which was inserted longitudinally into the muscle fibre. 2. When the excitatory nerve was stimulated, a transient feed-back current flowed inwardly through the membrane. This current was called the excitatory junctional current (e.j.c.). 3. Reversal potentials were determined by extrapolating the e.j.c.s measured at different membrane potentials. They were about 10-20 mV positive with respect to the bath solution (11-5 +/- 1-2 mV, mean +/- S.E.). 4. The reversal potential for the iontophoretically applied glutamate was identical with that for the e.j.c. 5. In hypertonic solutions, the reversal potentials for e.j.c. and glutamate became more negative. 6. When the sodium concentration of the bath solution was decreased, the reversal potential became more negative. 7. When the chloride and potassium concentration were altered, little, if any, change was observed in the reversal potential. 8. It was concluded that the e.j.c. was carried mainly by sodium ions. Contribution of other ions, possibly calcium ions, was discussed.

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

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.

Studies on Carotenoprotein In Aquatic Animals : Ill. The Relationship between Blue and Purple Carotenoproteins in Crayfish (Cambarus clarkii) Exoskeleton

アメリカザリガニ甲殼から得た青色カロチノプロテイン(以下Bと略記)と紫色カロチノプロテイン(以下Pと略記)のアポ蛋白質の分析結果より,それぞれのアポ蛋白質は異ったサブユニットで構成されていることを明らかにした. 約10万の分子量をもつBは尿素の存在により分子量約5万のPに変化する。Bは,ある条件下で,補欠分子族のカロチノイド含量が減少することにより,Pおよびカロチノイドを含まない蛋白質に解裂し,最終的には縮合して赤色カロチノプロテインを生ずる.甲殼内においてもBとPは相互変換が起りうると考えた.