Nov 5, 1990 - Abstract--I. The effect of eyestalk ablation on preadults of Cullinecfes similis exposed to a constant salinity (3%) and to simulated tidal changes ...
Camp.&‘&em. Physiol.Vol. IOOA, No. I, Pp. 7-O. 1991 Printed in GreatBritain
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EFFECT OF EYESTALK ABLATION ON OXYGEN CONSUMPTION OF CALLINECTES SIMILIS EXPOSED TO SALINITY CHANGES CARLOSROSA%* CECILIAVANEGAS,*GUILLERMINA ALCARAZ*
and FERNANDOD~Az~ *Laboratorio de Ecofisiologia, Depto. de Biologia, Fat. de Ciencias, UNAM 04510, MCxico D. F. Fax: 548-8186; TAcuario, Dept. Biologia, Fat. de Ciencias, UNAM 04510, MCxico D. F. (Received 5 November 1990) Abstract--I. The effect of eyestalk ablation on preadults of Cullinecfes similis exposed to a constant salinity (3%) and to simulated tidal changes in salinity (30-11 to 30%0) were measured. 2. In constant salinity, crabs showed a persistent respiratory rhythm, with a maximum oxygen consumption during the day. Under these conditions, ablation significantly increased the respiratory rate but not the rhythm. 3. In variable salinities, the highest respiratory rates occurred in salinities of 11 and 16%0during the night. In these crabs, ablation of eyestalks and subsequent injection of eyestalk extracts did not alter the respiration rate rhythm. 4. The circadian rhythm is controlled by the periodicity of environmental changes instead of the influence of eyestalk hormones. 5. Regulation of metabolism in C. simih associated with osmoregulation involves other neurosecretory
INTRODUCTION
(Diaz et al., 1989; Heit and Fingerman, 1975; Kleinholtz, 1976; Silverthorn, 1975a). However, this line of research has been carried out mainly with euryhaline organisms such as C. maenas, C. sapidus and U. pugilator, that are capable of regulating their inner medium. CaNinectes similis is a crab species that lives mainly in marine environments associated with coastal lagoons. According to Engel (1977), this is a euryhaline species that is limited to I$%, below which it dies (Rosas, 1989). Rosas et al. (1989) found that C. simih does not significantly modify its metabolic rate when exposed to a diluted medium, and this indicates that it uses little or no energy to carry out its inner medium regulation processes. This type of response has been associated with that of osmoconform stenohaline organisms (Kinne, 1971). This research has focused on the role of eyestalk hormones in the regulation of the respiratory metabolism of C. similis preadults when exposed to salinity changes. The objective was to determine whether the hormonal regulation mechanisms reported for euryhaline species (C. maenas, U. pugilator and C. sapidus) occur in other limited euryhaline species which, like C. similis, colonize estuarine environments (Rosas, 1989).
The role of the neuroendocrine system in the regulation of the ionic and osmotic balance in the decapod Crustacea is evident. Zatta (1987), reported that hypo-osmotic conditions increase the concentration of dopamine, noradrenaline and serotonin in the hemolymph of Carcinus maenas. Kamemoto and
Oyama (1982), reported that a preparation of the pericardial organ produced an increase in the flow of Na + and AMPc in the perfused gills of C. maenas and Callinectes sapidus. In these sense the pericardial organ has been proposed as the neurosecretory source of biogenic amines in Crustacea. Ablation of eyestalks of Procambarus clarki acclimatized to diluted media produces a decrease in the total concentration of salts, and that effect is reversed by injection of eyestalk extract (Kamemoto et al., 1966). The blood Na+ of Uca pugilator with ablated eyestalks increased after injection of eyestalk extract (Heit and Fingerman 1975). These authors concluded that a hormonal factor inhibits the loss of blood Na+ in hyposmotic sea water. The respiratory metabolism has been considered a good indicator of the general physiological state of Crustacea, as it includes the energetic needs for many metabolic processes, among them osmotic regulation (Findley et al., 1978). This is due to the fact that most of the regulation processes that take place in the inner medium require energy demanding processes, such as activity of conveying enzymes, transportation of aminoacids, cardiac activity and movement of the scaphognatite. It is possible to think that these processes change oxygen consumption in eyeless organisms exposed to diluted salinities, since hormones intervene in the activation of many of these processes
MATERIALSAND METHODS
Forty C. similis preadults (20.3 f 3.0 g wet weight) were captured in the southern part of Tamiahua lagoon, Veracruz, situed in the central portion of the coast of the Gulf of Mexico, they were kept in glass aquaria with 60 1 of constantly aereated lagoon water at 30 + 1% and 27 f 1°C. The specimens remained for 24 hr under such conditions before the experimental phase was commenced. All animals were in intermolt stage. 75
CARLOS
76
ROSM et al. Table 1. C. similis.Oxygen consumption maximum, minimum and metabolic amplitude obtained from circadian rhythms. Values in mgO,/hr/gdw. A = crabs exposed to constant salinity of 3% (IV = 20). B = crabs exposed to salinity changes (N-= 20)
After this time, both eyestalks were totally ablated in a group of 20 crabs. In order to avoid the stress produced by the ablation, the second eyestalk was eliminated 24 hr after the first. Eyestalk ablation was followed by cauterization by heat to minimize mortality due to loss of haemolymph or to infection. Forty-eight hours after ablation all specimens were placed individually in 3 1respiration measuring chambers to evaluate the effect of ablation on the respiratory metabolism associated with inner medium regulation. Whole and eyestalkless crabs were exposed to constant salinity (30 f 1%) as well as to the effect of an artificial tide with a change in salinity every 3 hr during a 24 hr cycle, according to the procedure used by Vanegas et al. (1988). Salinity was changed in the chambers from 30?&1,to 26, 21, 11, 16, 21, 26 and 30%. Closed respiration measuring chambers were used to determine oxygen consumption. The chambers were kept closed for 30 min to avoid alterations in the metabolic rate of the crabs due to a decrease in oxygen concentration and the accumulation of excretion products. These results were corrected with data from control chambers for each experimental group without crab. After this, water was changed, whether with constant salinity or with a new salinity concentration. Once this experiment was finished, an eyestalk extract was prepared as has been proposed by Skinner (1985). Eyestalks were macerated in 80% alcohol to obtain a homogenized product, after which the alcohol was allowed to evaporate. The product was suspended in crab ringer solution of which 100 ~1 were injected into the base of the fifth pair of legs of the operated specimens. Normal crabs were injected with 100~1 of crab ringer in the same arthrodial membrane
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*Crabs injected with crab ringer. tCrabs injected with eyestalk extract. %P < 0.05.
in order to create the same stress conditions caused by handling. Oxygen consumption was measured once more, as has been described after which the organisms killed and dehydrated at 60°C until constant weight was attained. Results were expressed in mg OJhr/g dw. In order to compare the metabolic rate in normal and ablated organisms, the metabolic amplitude of routine metabolism was calculated (Diax-lglesia, 1976) from the difference between the maximum and minimum oxygen consumptions obtained in each 24 hr cycle (Table 1). The non parametric Kruskal-Wallis test was used to find out the level of significance of the oxygen consumption
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Fig. 1. Oxygen consumption rhythm (mg O,jhr/gdw) of C. similis exposed to constant salinity (30%0). A = normal animals. B = Normal animals injected with crab ringer. Vertical bars represent the 95% confidence limits to the median.
Effect of eyestalk ablation on crustacean oxygen consumption Bilaterally stalkless specimens
values that were obtained for each experimental group through the 24 hr cycle. The non parametric Mann-Whitney
test was also used for comparisons between experimental groups (Zar, 1974). The median f95% interval of confidence was used to measure the central tendency of the results. RESULTS Normal specimens
Normal specimens were considered as a control group, in constant as well as in changing salinity. The variation in oxygen consumption in normal crabs exposed to constant salinity is shown in Fig. 1. These organisms present a unimodal circadian rhythm with greater oxygen consumption during the day than at night (P < 0.05) with maximum values at 0700 hr (0.24 mg O,/hr/g) and at 0900 and 2300 hr (0.24 mg O,/hr/g) respectively. No significant differences were recorded between metabolic levels of both, before and after having been injected with crab ringer control groups (P > 0.05) (Table 1). Figure 2 shows oxygen consumption in normal crabs exposed to changing salinity. It is clear that unlike organisms exposed to constant salinity, these crabs consumed more oxygen during the night (P < 0.05) when salinity was reduced to 16 and ll%a. No significant differences were found between the maximum oxygen consumption levels of the specimens before and after having been injected with crab ringer (P > 0.05; Table 1).
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Figure 3 shows oxygen consumption in stalkless specimens exposed to constant salinity. A remarkable difference appears between oxygen consumption in operated specimens and that of those that were operated and were injected with eyestalk extract. In operated specimens (Fig. 3A) a unimodal circadian rhythm with a maximum oxygen consumption during the day of 0.42 mgO,/hr/g at 1600 hr (P < 0.05); Table 1) can be observed as in whole crabs. This value turned out to be 1.6 times larger than that obtained for whole specimens (P < 0.05). In contrast, when this group was injected with eyestalk extract (Fig. 3B), the metabolic rate decreased to the levels registered in normal organisms injected with crab ringer (P > 0.05) without a definite circadian rhythm. Although not significant, a maximum oxygen consumption of 0.32 mg Or/hr/g was found to occur at 0600 hr in this group of crabs (Table 1). Figure 4 shows the oxygen consumption of C. similis after being operated and exposed to a simulated tidal rhythm of salinities between 30 and 11%. Operated specimens (Fig. 4A) presented a similar rhythms that observed in whole organisms: a unimoda1 circadian rhythm with a maximum level of oxygen consumption at night with a salinity of 16% (0.36 mgO,/hr/g; P < 0.05; Table 1). It was also observed that after this maximum level oxygen consumption decreased and remained stable throughout the other salinities. When this group of organisms
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Fig. 2. Oxygen consumption rhythm (mg O,/hr/g dw) of C. similis exposed to salinity changes. A = normal animals. B = normal animals injected with crab ringer. Vertical bars represent the 95% confidence limits to the median.
CARLOS ROSM et al.
78
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Fig. 3. Oxygen consumption rhythm (mg OJhr/g dw) of eyestalkless C. similis exposed to constant salinity (30?60).A = eyestalkless crabs. B = eyestalkless crabs injected with eyestalk extract. Vertical bars represent the 95% confidence limits to the median.
was injected with eyestalk extract and exposed to the same changing environment, maximum oxygen consumption was also observed between 11 and 21% during the night (Fig. 4B). Metabolic
amplitude
Changes in salinity produced a significant increase in metabolic amplitude in normal specimens compared to the metabolic amplitude obtained for specimens exposed to constant salinity. That metabolic amplitude was 2-2.5 times larger (P < 0.05). In contrast eyestalk ablation produced an increase in metabolic amplitude when specimens were in constant salinity compared to the amplitude registered for crabs exposed to the changing environment (P < 0.05). Thus, metabolic amplitude in operated specimens exposed to 30%0 was 1.83 times larger than that registered for operated organisms in the changing environment (Table 1). However, when these organisms were injected with eyestalk extract a sudden decrease in metabolic amplitude was registered, which reached levels close to those found in normal specimens exposed to constant salinity (P > 0.05). It is to be noted that the metabolic amplitude and the circadian behaviour of metabolism in animals exposed to changing salinity was not altered by the injection of crab ringer, ablation or injection of eyestalk extract (Table 1).
DISCUSSION
C. similis presented a daytime unimodal circadian rhythm when placed in constant salinity. Periodicity was not altered by injection of crab ringer or by ablation of eyestalks. Ablation produced an increase in metabolic amplitude which indicated a general alteration of the metabolism. These responses have been associated to changes produced in the whole metabolism by ablation of eyestalks. Brito and DiazIglesia (1987) and Diaz-Iglesia et al. (1987) worked with Panulirus argus and found that ablation produced several metabolic alterations among which is hypoglycemia, a decrease in glycogen and an increase in lipids in the hepatopancreas as well as an increase in glycogen in the muscles. These alterations were associated to an increase in oxygen consumption and growth and agree with those obtained by Madhyastha and Rangneker (1976) for the Varwla iitteruta crab and by Mauviot and Caste11 (1976) for the Homarus americanus lobster. These authors agree that such alterations are produced by the elimination of the moult inhibiting hormone with the consequent acceleration of the metabolism of growth. Silverthom (1975a and b) has proposed the existence of a respiratory repressing hormonal factor in the eyestalks, which, when eliminated by ablation allows the respiratory stimulating hormone to rush the respiratory rate. Thus, the general metabolic
Etfect of eyestalk ablation on cm&man oxygen ~ns~ptioo
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Fig. 4. Oxygen ~nsumption rhythm (mg O&r/g dw) of eyestalkless C, simiiisexposed to salinity changes. A = eyestalkless crabs. B = eyestalkless crabs injected with eyestalk extract. Vertical bars represent the 95% confidence limits to the median.
changes as well as the presence of respiratory regulating hormones, caused oxygen consumption of C. s~j~~ to be modified in magnitude when organisms in constant salinity were submitted to eyestalk ablation. As for the rhythm, it was observed that neither ablation nor eyestalk extract injections modified the unimodal circadian rhythm measured in a 24 hr cycle. Vanegas er al. (1988) have demonstrated the existence of a persistent unimodal circadian rhythm in C. similes preadults, with a maximum activity in oxygen consumption during daytime. In accordance with the results obtained in our investigation, this rhythm is controlled by daily variations in salinity, temperature, photoperiod, tides, winds, etc. (Natarajan, 1989), and not by hormonal changes. As for the eyestalk hormones possibly taking part in inner medium regulation, our results make it clear that this neurosecreting complex has no role in the process. This was evident when ablation produced no alterations either in the rhythm of oxygen consump tion or in the level of routine metabolic amplitude. Concerning the circadian rhythm that was observed, it can be noticed that, specimens in the changing regime presented their maximum activity during the night when salinity varied between 11 and 16% unlike the organisms in constant salinity. C. similis is a limited euryhaline organism that can regulate its inner medium for short spells of time
(24 hr) in salinities up to 11% (Engel 1977; ROW 1989). If we take into account that such an inner medium regulation requires an energy investment (Findley et al., 1978), it is possible to think that an increase in metabolic rate is related to the energy involved in inner medium regulation at the above mentioned salinities. These results differ from those obtained by Heit and Fingerman (1973, who suggested that the eyestalk hormonal factor that prevents Na+ loss and stimulates the Na+-K+ ATPase in diluted media, would perceptibly increase the metabolism of specimens. In this research, eyestalk ablation and injection of eyestalk extract produced no alteration in oxygen consumption in the C. similh specimens exposed to changing salinity. In relation to this, Zatta (1987) has pointed out that dopamine (DA), noradrenaline (NA) and serotonin (SHTP) are modified when Carcinus maenas is exposed to 50% sea water. These hormones, secreted by the pericardial organ, increase Na+ flow, AMPc level in gills and the cardiac rate, and also modulate water flow through the scaphognatite. if we consider that energy from metabolism is invested in such processes, increase in oxygen consumption occurs if there is a change in the concentration of hormones in specimens that are in diluted media (Araujo and Castro, 1985). It would therefore be possible to establish that the eyestalk hormones in C. simiib do not play an
CAtu.0s Rm
80
important role in osmotic regulation but the pericardial organ hormones do intervene in the regulation of this process. This neuroendocrine regulation scheme in osmotic balance might be applied to crustacea which, like C. similis, are limited euryhalines in colonization processes of estuarine environments (Rosas et al., 1989). Considering this and the fact that evidence has been found of the existence of hydromineral balance regulating hormones in pericardial organs as well as in the eyestalks of euryhaline organisms such as C. sap&s, Uca pugilator and C. maenas, we suggest that the function of the pericardial organ as a producer of inner medium regulating hormones in marine or limited euryhaline species appeared first in crustacea such as C. similis that colonise estuarine ecosystems. Acknowledgements-This
research was supported by the Division of Postgraduates of the Faculty of Sciences of Universidad National Autbnoma de M&co. We are grateful to Fernando Biickle-Ramirez and Rafael VillalobosMolina for constructive comments and to M. en C. Andrea Raz-Guzman Macbeth for English corrections.
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