seasonal rhythm of red pigment concentrating hormone in the crayfish

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CHRONOBIOLOGY INTERNATIONAL, 14(6), 639-645 (1997) SEASONAL RHYTHM OF RED PIGMENT CONCENTRATING HORMONE IN THE CRAYFISH Leonardo Rodriguez-Sosa,' Ma. Teresa de la Vega: Paula Vergara: and Hugo Arkchigal 'Divisi6n de Estudios de Posgrado e Investigacibn, Facultad de Medicina, UNAM, MBxico, D.F. 'Departamento de Fisiologia, Biofisica y Neurociencias, Centro de Investigacih y de Estudios Avanzados, IPN, MCxico, D.F. ABSTRACT The content of red pigment concentrating hormone (RPCH) in the eye- stalk of the crayfish Procambarus clarkii varies seasonally, with maximum values during the summer months and the lowest values in winter. The re- sponsiveness of tegumentary chromatophores to synthetic RPCH varies con- currently. (Chronobiology International, 14(6), 639-645, 1997) Key Words: Crayfish-Seasonal rhythm-Red pigment concentrating hor- mone-Circadian rhythm. INTRODUCTION Seasonal cycles in crustaceans have been known for some time (1,2) and affect a number of physiological functions, such as molting (3,4), reproduction (5-7), locomotion (8,9), metabolic activity (7,10,1 l), and behavioral responses to environmental stimuli (12). There is, however, scarce information about the neurobiological substrate of sea- sonal rhythms. A seasonal rhythm of visual responsiveness has been described in Nephrops nor- vegicus (12), and various factors may contribute. In some crustacean species, the spectral sensitivity of retinal photoreceptors displays a seasonal change (1 3,14). Retinal shielding pigments may also play a role; they are known to modulate the light admittance to the eye and, consequently, the responsiveness to light (15-18). Various neurotransmitters and Received February 14. 1997; returned for revision April 16, 1997. Address correspondence to: Leonardo Rodriguez-Sosa, Facultad de Medicina, Divisi6n de Estudios de Posgrado e Investigacih, Unidad de Posgrado ler, Piso Circuit0 Interior C.U., Mtx- ico, 04S10, D.F. 639 0 1997 International Society for Chronobiology Chronobiol Int Downloaded from informahealthcare.com by Michigan University on 10/31/14 For personal use only.

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CHRONOBIOLOGY INTERNATIONAL, 14(6), 639-645 (1997)

SEASONAL RHYTHM OF RED PIGMENT CONCENTRATING

HORMONE IN THE CRAYFISH

Leonardo Rodriguez-Sosa,' Ma. Teresa de la Vega: Paula Vergara: and Hugo Arkchigal

'Divisi6n de Estudios de Posgrado e Investigacibn, Facultad de Medicina, UNAM, MBxico, D.F.

'Departamento de Fisiologia, Biofisica y Neurociencias, Centro de Investigacih y de Estudios Avanzados, IPN, MCxico, D.F.

ABSTRACT

The content of red pigment concentrating hormone (RPCH) in the eye- stalk of the crayfish Procambarus clarkii varies seasonally, with maximum values during the summer months and the lowest values in winter. The re- sponsiveness of tegumentary chromatophores to synthetic RPCH varies con- currently. (Chronobiology International, 14(6), 639-645, 1997)

Key Words: Crayfish-Seasonal rhythm-Red pigment concentrating hor- mone-Circadian rhythm.

INTRODUCTION

Seasonal cycles in crustaceans have been known for some time (1,2) and affect a number of physiological functions, such as molting (3,4), reproduction (5-7), locomotion (8,9), metabolic activity (7,10,1 l), and behavioral responses to environmental stimuli (12). There is, however, scarce information about the neurobiological substrate of sea- sonal rhythms.

A seasonal rhythm of visual responsiveness has been described in Nephrops nor- vegicus (12), and various factors may contribute. In some crustacean species, the spectral sensitivity of retinal photoreceptors displays a seasonal change ( 1 3,14). Retinal shielding pigments may also play a role; they are known to modulate the light admittance to the eye and, consequently, the responsiveness to light (15-18). Various neurotransmitters and

Received February 14. 1997; returned for revision April 16, 1997. Address correspondence to: Leonardo Rodriguez-Sosa, Facultad de Medicina, Divisi6n de

Estudios de Posgrado e Investigacih, Unidad de Posgrado ler, Piso Circuit0 Interior C.U., Mtx- ico, 04S10, D.F.

639

0 1997 International Society for Chronobiology

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640 RODRiGUEZ-SOSA ET AL.

modulators have been postulated in the control of the position of retinal shielding pig- ments (19). The octapeptide red pigment concentrating hormone (RPCH) is one such agent. Besides its role in inducing the retraction of pigment granules in tegumentary erythrophores, it promotes the retraction of distal retinal pigment granules, which in- creases the photon flux in the retina, thereby enhancing the retinal response to illumina- tion (20). The RPCH content in the eyestalk has been determined (21) and has been shown to vary in a circadian manner (22), but no study has been made of possible changes of RPCH throughout the year. The purpose of this paper is to present evidence for the existence of a seasonal rhythm of RPCH content in the crayfish eyestalk and concurrent changes in the responsiveness to RPCH.

MATERIAL AND METHODS

The experiments were conducted in adult crayfish Procambarus clarkii (Girard) of either sex. Animals with carapace lengths between 9 and 11 cm were selected. This size corresponds to adult specimens between one and two years old. The animals were col- lected from Rio Conchos, Chihuahua, MCxico, about 1 4 weeks prior to the experiments and kept under laboratory conditions, in 12: 12 light-dark cycles, and at room tempera- tures close to 20°C. Usually, groups of three animals were sacrificed for a given sample. The eyestalks were removed, and the exoskeleton, connective tissue, and muscles sur- rounding the optic peduncle were excised by microdissection.

The RPCH was purified from the samples by high-performance liquid chromato- graphy (HPLC). Three methods were used to quantify RPCH, as previously described (21,22): (i) by the magnitude of the fluorescence detected by HPLC; (ii) by an immu- noenzymatic method (enzyme-linked immunosorbent assay, ELISA) using an antibody raised in our laboratory against synthetic RPCH-Tyr, which recognized the 4-5 residues at the amino terminal; and (iii) by a bioassay, by topical application on isolated flaps of tegument, containing a large population of erythrophores. Between them the three meth- ods covered a range of RPCH sensitivity, detecting from lo-" to

The red pigment concentrating effect was assessed by microscopic observation of each preparation at 15-minute intervals during a 60-minute span. Pigment dispersion was rated in five stages (Stages 1-5, from minimum to maximum dispersion) at different times before and after RPCH application. The potency to induce pigment concentration was calculated as the index of concentration (IC), which was calculated from the equa- tion:

M.

IC = (Cis + C,, + C45 + Ca - El5 + E30 + E45 + Em) C and E are, respectively, the average dispersion of the red pigment in the control and experimental epithelia at 15-minute intervals following application. The synthetic RPCH used in our experiments was purchased from Peninsula Laboratories (Belmont, CA). The experiments were carried out during the daytime and after light adaptation, so that the control value was a fully dispersed pigment. The experiments were performed at a con- stant temperature of 22OC. A more detailed description of the procedure is given in Refs. 21 and 22.

RESULTS During the three years of sampling (1991-1994), a consistent pattern of RPCH

content was detected in the eyestalk. Figure 1 shows the monthly average content for the

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SEASONAL RHYTHM OF RPCH IN THE CRAYF'ISH 641

FIGURE 1. Red pigment concentrating hormone (RPCH) content in the crayfish eyestalk (ordi- nate, ndeyestalk) during the year. Bars indicate average values and standard deviations at monthly intervals, and the inset shows the distributions after pooling the data on quarterly basis. The num- ber of samples in the pool is indicated above the bars in the inset.

three years. A consistent low value during the winter months was followed by an abrupt increase from March to April, when the RPCH content doubled in one month. This high level was maintained during springtime, and a further increment occurred during sum- mer. The highest value in the year was attained in July (6.1 ng/eyestalk). The content remained high during autumn and showed a precipitous decline in early winter, dropping from 5.7 ngleyestalk in December to 1.7 ng/eyestalk in January, with the lowest level of 1.5 @eyestalk in February. The inset of Fig. 1 presents data pooled for the four quarters. As seen, RPCH content during summer (5.5 ng/eyestalk) and autumn (5.6 ng/eyestalk) was nearly three times larger than in winter (1.8 ng/eyestalk) and almost twice the amount detected in spring (2.9 ng/eyestalk).

Seasonal variation in the sensitivity of tegumentary chromatophores to RPCH was investigated in a monthly study of the responses to synthetic RPCH applied in vitro to isolated flaps of tegument, such as those used when bioassaying RPCH. Both the final stage of pigment retraction and the time course of the response were determined for three concentrations of RPCH (lo4 M, lo-' M, and lo-' M). As seen in Fig. 2, the RPCH-

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642

5-

4-

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RODRiGUEZ-SOSA ET AL.

D CQNTROL RPCH ~ O - ~ M RPCH 10dM

0 RPCH M

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TIME (min.) FIGURE 2. Time course of red pigment concentration induced by red pigment concentrating hormone (RPCH) applied at three doses as indicated. A, data from December to February. B, data from March to May. C, data from June to August. D, data from September to November. Ordinate indicates the stage of pigment dispersion (from a minimum of 1 to a maximum of 5). Symbols indicate average values and standard error (n = 40). Inset at top shows the extent of maximum RPCH-induced pigment concentration at the three doses tested in each of the four quarters, as indicated (see “Material and Methods” section).

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SEASONAL RHYTHM OF RPCH IN THE CRAYFISH 643

induced retraction of tegumentary pigment, expressed as an index of concentration, and its time course varied depending on the time of year. Again, the winter months were those of the lowest responsiveness. The maximum winter response was smaller than in the rest of the year at all RPCH doses tested. The inset at the top of Fig. 2 shows the dose-response curves for the maximum retractions induced by the three RPCH doses on a quarterly basis. At all doses, but most noticeably at the lowest, the response was greater in summer and autumn than in winter.

DISCUSSION

The seasonal rhythm described here is the first of this nature documented for a crustacean neurohormone. It is interesting that the highest values of endogenous, basal RPCH content in the eyestalk, and tegumentary responsiveness to extrinsic RPCH, were all found in summer. This is consistent with our previous finding that the amplitude of the rhythm of RPCH content in the crayfish eyestalk under light-dark cycles is greater than in either continuous darkness or constant illumination (2 l), thus suggesting that the alternation of light and darkness plays a role in stimulating RPCH secretion. The close temporal correlation of tegumentary responsiveness to RPCH with the rhythm of RPCH content deserves further analysis. Most examples of rhythmic variations in the number and affinity of receptors to hormones and neuroactive substances have been reported in vertebrates (23-25). There is no evidence of rhythmic changes of hormone receptors in crustaceans. It is also pertinent to consider that, in our experiments, no specific control was made of changes in connective tissue distribution, which might also change during the year, resulting in differences of permeability to the applied substances.

Given the functional role of RPCH in the control of retinal sensitivity (20), it is conceivable that a higher responsiveness of the retina during the summer months might contribute to the enhancement of photomotor reactivity to light-darkness cycles, thus enabling the animals to adapt to wider ranges of daily light intensity fluctuations. Sum- mer is also the time of year when, in other crustacean species, the mobility of the retinal shielding pigments is greater (12) and the locomotor activity is higher (8,9).

The responsiveness to some putative neurotransmitters and modulators also appears to peak in summer. Such is the case of the hyperglycemic response induced by Shy- droxytryptamine in the crab Curcinus menus (26) and the activation of a voltage-depen- dent calcium current in the crayfish muscle induced by proctolin (27). The depression of transmitter release after repetitive stimulation in the neuromuscular junction of Procumb- nrus clarkii is smaller in summer than in winter (28), and the summer crayfishes appear to have more synaptic vesicles and mitochondria in the varicosities at the motor terminals (29). These cyclical changes may contribute to a greater motor activity and responsive- ness to environmental stimuli in summer and autumn.

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