y. exp. Biol. (1978), 7*, 57-75 5 7With 7 figures

Printed in Great Britain

ASPECTS OF THE PHYSIOLOGY OF TERRESTRIAL LIFEIN AMPHIBIOUS FISHES

III. THE CHINESE MUDSKIPPER PERIOPHTHALMUS CANTONENSIS

BY MALCOLM S. GORDON,* WILSON W.-s. NGf ANDALICE Y.-w. YIP}

• Department of Biology, University of California, Los Angeles,f Department of Extramural Studies, University of Hong Kong, Hong Kong and

X Po Shing Building, Yuen Long, Hong Kong

(Received 23 May 1977)

SUMMARY

1. Aspects of the physiological adaptations for terrestrial life possessed bythe Chinese mudskipper fish, Periophthalmus cantonensis have been studied.

2. The basic amphibious features were comparable to those foundearlier in the East African mudskipper species, Periophthalmus sobrinus. Thefish can survive for up to 2 | days out of water, under moderate environ-mental conditions, and did not drown in aerated water. Durations of volun-tary periods out of contact with water, in nature, were short. Evaporativewater loss rates while out of water were relatively low, substantially belowthose of frogs of much larger size. The 'diving syndrome' was absent,metabolic rates, heart rates, and blood lactic acid levels all being unaffectedby shifts of fish between water and air.

3. During slow desiccation of intact fish, water was lost from the internalorgans in the following sequence (from greatest to least proportional waterlosses): heart, blood, white muscle, brain, liver. Rapid desiccations withdrewwater from the blood and heart, leaving hydration levels of other organsunaffected.

4. Metabolic rates were only moderately sensitive to temperature in therange io°-2o °C (Q10 ^ 1-5), but were highly temperature sensitive in therange 2O°-3O cC (Q1O ^ 2-6). Measurements of routine metabolic rates offish in water indicate that active metabolism at 20 °C can reach at least5 times the standard rate.

5. Measurements of blood levels of ammonia and urea, and of ammoniaand urea excretion rates, showed that starvation for 9-5 days did not affectblood levels of these compounds, but did reduce urea excretion rates by 40%below rates for recently fed fish. Ammonia excretion was unaffected.

6. Similar measurements on fish starved for 9-5 days, then kept out ofwater for 16-5 h, then returned to water, indicated that waste nitrogenproduced by fish out of water was retained in their bodies until they returnedto water. Fish out of water produced waste nitrogen 40 % less rapidly thanfish in water, and strongly shifted toward ureotelism. These experiments,however, did not permit fish to retain and renew water in their mouths, asthey would have done in nature.

58 M. S. GORDON, W. W.-S . NG AND A. Y.-w. YIP

7. The water in mudskipper burrows was found to be anoxic or nearly so.Mudskipper tolerance for hypoxia was, however, limited. Fish became inertat dissolved oxygen levels of about 20% saturation.

INTRODUCTION

How much of the biochemical, physiological, and behavioural machinery whichmust have helped form the basis for the evolutionary transition of the vertebratesfrom aquatic to terrestrial environments might have pre-existed in the fishes?Might there have been only one, or were there perhaps several, general patternsof functional adaptation involved in this transition?

Studies of the living lungfishes (Dipnoi), considered the group of modern fishesphylogenetically closest to the ancestral, completely extinct, rhipidistian cross-opterygians, have provided plausible partial answers to these questions (Thomson,1969, 1971)- Additional partial answers have also been obtained from studies ofliving amphibious fishes, especially those forms least structurally specialized for lifeout of water. This latter group seems most likely to demonstrate both the maximumextent to which fishes are capable of living terrestrially, and the diversity of responsepatterns used to solve the problems involved (Gordon et al. 1969; Gordon, Fischer& Tarifeno, 1970; Gordon, 1970; Ebeling, Bernal & Zuleta, 1970; Graham, 1970,1973, 1976; Tamura, Morii & Yuzuriha, 1976).

The mudskippers of the family Periophthalmidae, especially those species nowrecognized as distinct, but previously included in the widely distributed speciesPeriophthalmus koebeuteri (Koumans, 1953), are among the most terrestrial of knownamphibious fishes. They usually live in the upper reaches of the intertidal zone andoften spend more than 90% of their time out of contact with liquid water. They arephysiologically well adapted to terrestrial life (Gordon et al. 1969; Tamura et al.1976).

The present paper provides information comparable to that presented by Gordonet al. (1969) for an East Asiatic species belonging to this group, P. cantonensis (Kou-mans, 1953; Milward, 1974). It expands in several respects the scope of the previouswork on both this species (Tamura et al. 1976) and the group. Its most importantnew features include: (i) investigations of aspects of internal distributions of waterand solutes in mudskippers subjected to desiccation stresses: (ii) determination oftemperature and activity effects on metabolic rates; (iii) quantitative determinationof the nature of changes in patterns and rates of waste nitrogen production andexcretion resulting from periods of time out of water; and (iv) study of responsesof mudskippers to anoxic conditions comparable to those encountered in theirburrows.

MATERIALS AND METHODS

Mudskippers ranging in size from recently metamorphosed post-larvae to adultsweighing up to about 5 g were usually abundant on mangrove forest intertidal mudflats in several localities in the northeastern parts of the New Territories, Hong Kong.Average maximum body weight was near 3 g. Adequate numbers of fish for ex-perimental uses were readily caught, during the period September 1971 to June

Terrestrial life in amphibious fishes. Ill 59

1972, in small dip-nets during low tide periods at night. All experimental work wasdone at the Marine Science Laboratory, Chinese University of Hong Kong, Shatin.

Fish to be used promptly, in short-term experiments, were maintained in thelaboratory in 40 1 capacity plastic aquaria with up to about 20 fish per aquarium.The aquaria were filled at intervals of 1-2 days to depths of 2-3 cm with sea waterfrom the adjacent Tolo Harbour (salinity normally 29-31 %o, except for variableperiods of dilution following heavy rains; Trott, 1972). Rocks were provided forthe fish to climb on. No attempt was made to separate the sexes. These fish werenormally not fed, unless conditions prevented their use within 7-10 days followingcapture. Laboratory air temperatures varied with ambient temperatures. Diurnalranges were usually near 5 °C. Seasonal ranges were from near 3 °C (night tempera-tures for a period of about 3 weeks in January-February) to about 30 °C (clear daymid-afternoon temperatures from May-October).

A reserve supply of experimental fish, also a group offish used solely for behaviouralobservations, was maintained in a 3-5 m diameter circular concrete outdoor tank.This tank had smooth concrete walls 1 m high. The bottom of this tank was abouttwo-thirds filled to a depth of 15-20 cm with mud from a nearby mangrove forestwhich supported a natural mudskipper population. The remainder of the bottomof the tank was filled with slowly flowing Tolo Harbour sea water to a depth ofabout 10 cm. The numbers of fish in this artificial mud flat varied at times from onlya few to near 100. Some individual fish lived in this tank for at least 6-8 months.Fish in this tank (also fish in aquaria for long periods) were fed on broken up bitsof fresh marine clams and snails at least twice each week.

The following observations and experiments were carried out, using intact fish(calendar months during which the work was done indicated for each topic):

Survival in water and air (October). In water: a group of six fish (2-3 g body weight)was placed in a 40 1 plastic aquarium which was then filled three quarters full withsea water. A piece of plastic fly screen was mounted inside the tank, several cm belowthe water surface, so that the fish had no access to air. The water was aerated con-tinuously with a small air pump and diffuser stones, the stones placed above thescreen. Water was changed at intervals of several days by siphoning, care beingtaken to avoid giving the fish any access to the surface. Water temperature was22-24 °C. Survival was monitored for 6 days. In air: in a moist chamber as in Gordonet al. (1969), except that 12 fish were used, in two groups of six each, segregated bybody weight (small fish, o-6-i-4 g; large fish 2*2-3-8 g). Air temperatures 22-28 °C.

Field behaviour with respect to contact with water (September-October). A populationof fish at a nearby mangrove area (Tai Po Kau) was observed for 30-60 min periodson clear, sunny days during low tide intervals over a period of 3 weeks in late Sep-tember and early October. During this time low tide intervals shifted from earlymorning to late afternoon. Individual fish were watched through binoculars, andstopwatch based records were made of the durations of periods completely out ofcontact with liquid water (from moment of last contact to moment of next contactby any part of the body). Data obtained are a mixture of multiple, sequential obser-vations on single fish and of single observations, each of a different fish.

Rates of evaporative water loss, and body temperatures (October). As in Gordon« al. (1969). Time courses of changes in body weight were determined for three

60 M. S. GORDON, W. W. -S . NG AND A. Y.-w. YIP

groups of six fish each. Individual fish were placed in screen-covered, tared plasticdishes. The groups were then exposed to the following conditions: (a) Still air inshade (initial fish weights i-S-3'2 g); (b) moving air (12-15 km h"1) in shade (initialfish weights i-i-i-8g); (c) moving air (5-12 km h"1) in full sun (initial fishweights I-I-I-6 g). A group of local fresh water frogs (Rana tigerina), weighingfrom 28 to 34 g, were also subjected to desiccation in still air and shade. Shade airtemperatures and relative humidities during experiments were: still air, shade (fish),23-24 °C, 40-55%; still air, shade (frogs), 24-260, 52-54%; moving air, shade,27-28 °C, 51-57%; moving air, sun, 30-310, 55-56%.

Distribution of water and some solutes in the bodies of fish subjected to desiccation(May-June). Groups of fish were desiccated to varying degrees both rapidly andslowly. Rapid dehydration experiments lasted 20 min; air moving at 12-14 km h"1

produced body weight changes at the average rate of 20% h"1. Slow dehydrationexperiments lasted up to 23 h; fish were kept in covered moist chambers and lostweight at the average rate of 2 % h-1. Air temperatures in all experiments 25-28 °C.At times of sampling fish were weighed, killed by blows to the head, and samplestaken of blood (by heart puncture) and major tissues (white trunk muscle fromabout half way along the body length; liver, heart, and brain). Blood plasma sampleswere analysed for freezing-point depression, sodium and chloride concentrations.Tissues samples were analysed for water contents (wet and dry weights). Samplehandling, sample sizes and analytical methods were all as used by Gordon (1965).

Metabolic rates (November-December). Two methods were used. For groups of3 or 4 post-larval fish (body weights of each near o-i g) and for individual smallfish (body weights o ^ - i ^ g), Scholander volumetric micro-respirometers were usedas described in Gordon et al. (1969). Measurements were made at temperatures ofio°, 20° and 30 °C, with chambers either dry or partly filled with sea water of thesalinity in which the fish had been living. Fish in the respirometer chambersnormally remained completely quiet for very long periods of time.

Other individual small fish, also six individual larger fish (body weights 3-O-4-7 g)were placed in sea water in sealable 435 ml volume glass jars, care having been takento eliminate all air bubbles before closing the jar. The jars with fish inside wereplaced in a 20 °C water bath and routine metabolic rates were determined. Watersamples of about 2 ml volume were taken at 1 h intervals for 5 h. Dissolved oxygencontents were determined using a Scholander dissolved gas analyser (Scholanderet al. 1955); analytical precision ± 0 1 ml oxygen 1-1. Sample volumes removed wereautomatically compensated for by addition of bubble-free air-equilibrated sea waterfrom reservoir syringes attached to each respirometer jar. Fish in these jars generallyrested quietly on the bottoms of the jars, but at irregular intervals for variable periodsof time they would make active swimming excursions around the jars. This is whyoxygen uptake rates measured in this way are termed routine metabolic rates.

Heart beat frequencies (May-June). By electrocardiogram, as in Gordon et al.(1970). Estimates of heart beat frequencies were made on eight fish of 2-3 g bodyweight, each subjected over periods of about 2 h duration to repeated forced sub-mersions and emersions between air at temperatures of 24-25 °C, and aerated seawater at temperatures of 26-27 °C- Results for individual fish were fairly consistent,but there was variability between fish. Electrodes were attached while the fish wen

Terrestrial life in amphibious fishes. HI 61

in air and dives were initiated 15-25 min later, after heart rates had declined frominitial excited levels and had stabilized.

Blood lactic acid concentrations (March-April). Colorimetrically, as in Gordon etal. (1970).

Ammonia and urea concentrations in blood (April-May). Whole blood samples takenby heart puncture. Analyses on duplicate 25 fi\ aliquots using urease and the microdiffusion method of Conway (Natelson, 1961, p. 440). Analytical precision for total(ammonia and urea) N, + 6 mg I"1. On the basis of results presented in Gordonet al. (1970), ammonia-N was assumed to be about 3% of this total.

Ammonia and urea excretion rates (January-March). As in Gordon et al. (1969),but modified. The sea water into which the experimental fish were placed wassteam autoclaved just prior to use, time only being allowed for it to cool to roomtemperatures of 19-23 °C. Water was replaced with new autoclaved water at ihintervals. This procedure eliminated all possibility of significant bacterial modificationof excreted waste nitrogen concentrations, or of pharmacological effects of bacterio-static agents added to the water. It also permitted hour by hour monitoring of ex-cretion rates, without concern for possible recycling by the fish of significant amountsof excreted wastes. Analyses on 250 ji\ water samples, by micro diffusion methodof Conway, as for blood.

Dissolved oxygen concentrations in water in mudskipper burrows (June). Samples ofwater at depths of 5-15 cm inside water-filled burrows were taken during low tideintervals by means of a 5 ml glass syringe fitted on its tip with a length of Tygonplastic tubing bearing on its distal end an aquarium air stone. Care was taken toeliminate all bubbles from the sampler prior to taking samples, and to avoid excessivedisturbance of the water in the burrow while sampling. Oxygen analyses were doneon the spot, immediately after sampling, using a Scholander dissolved-gas analyser(Scholander et al. 1955). Analytical precision +o-i ml oxygen I"1.

Tolerance for hypoxia (May). Seven fish were placed individually in sealable 130ml volume glass jars. The jars were filled with air equilibrated sea water, all bubbleseliminated, and they were sealed under water. The jars were kept at room tempera-tures of 24-27 °C. The fish were observed until they had ceased all breathing andswimming movements, and were unable to right themselves when their jar wasturned over. The jars were then opened and 2 ml water samples quickly taken fromnear the bottom. Dissolved oxygen concentrations were measured immediately,as for burrow oxygen concentrations. All fish quickly returned to normal activityfollowing return to better aerated conditions.

Statistical analyses. Wherever needed, estimates of degrees of statistical signifi-cance of differences between groups were made by analysis of variance (significantdifference means P ^ 0-05; highly significant difference means P ^ o-oi). Allregression calculations used standard programmes for linear least-squares regression.

RESULTS

Survival in water and air. All six fish which were kept in aerated sea water withoutaccess to air survived for 6 days. P. cantonensis does not drown in aerated sea water.NU1 12 fish placed in moist air chambers, without access to liquid water, survived for

3 EXB 7a

62 M. S. GORDON, W. W.-S. NG AND A. Y.-w. YIP

1oo6Z

20

10

20

a

8"o 10

oZ

10.20-11.1014 October

07.40-08.1522 October

6 8 10Time (min)

12

c 20o

a

8

oZ

10

10.45-11.3512 October

20

10

oZ

00

14.00-14.551 October

oo6

20

10

n

-

-

-

14.40-15.1028 September

rr^K^s i i i i i

0Time (min) Time (min)

Fig. i. Frequency distributions for durations of periods completely out of contact with liquidwater for Chinese mudskippers in early autumn on a natural mangrove forest mudflat in HongKong. Observations were made on clear, sunny days with light, variable winds, during the in-dicated time periods (which coincided with low tide intervals) on the indicated dates. Shade airtemperatures and relative humidities for the observation periods were, in sequence with clocktimes: 23°, 9 1 % ; 24°. 50%; 22°, 4 1 % ; 300, 73%; and 310, 63%.

22h. Mortalities then began to occur at irregular intervals. Times to death for theindividual small fish tested were 22, 23, 25, 30, 45 and about 60 h. Times to deathfor the larger fish tested were 23 (two fish), 26, 27, 30 and about 35 h. Percentagebody weight losses (presumably due to evaporation, even though the chamberatmospheres were presumed to be near water vapour saturation) at time of deathaveraged 36% in the small fish, 27% in the larger fish. P. cantonensis thus can readilytolerate at least a full day out of water, under shady, only moderately dehydratingconditions. Maximum tolerance for time out of water is at least z\ days.

Field behaviour with respect to contact with water. Frequency distributions fordurations of periods completely out of contact with liquid water, for undisturbedmudskippers actively moving spontaneously about in their own habitat, do not vary ii^

Terrestrial life in amphibious fishes. Ill

0 1 2 3 4Time (h)

Fig. a. Evaporative water loss (expressed as percentage change in body weight) in groups ofmudskippers in still air and shade (•), moving air (electric fan) and shade (©), and movingair (natural wind) and full afternoon sun (O). The fourth curve (A) presents similar data fora group of fresh-water frogs (Rana tigerina) in still air and shade. Six animals in each group.Plotted points are means for groups of observation*; vertical lines, ± 1 s.E.

statistically significant ways at times of day ranging from just after sunrise to lateafternoon (Fig. 1). This is the case in the face of bright sunshine, variable light breezes,and air temperatures varying from 22 to 31 °C.

The characteristic behaviour pattern at all times of day when the fish are notdisturbed or frightened by some external event is for most contacts with water to beshort in duration (usually less than 1 min), and restricted to a small part of the body,most often the mouth. The main stimulus for making contact with water appears to

fe the loss of the small volume of water carried in the mouth and gill chambers3-2

64 M. S. GORDON, W. W.-S . NG AND A. Y.-w. YIP

Changes in absolutevaluesf

i6o(39o-»- 550)90(150-* 240)

100 (140 -*• 240)

7 (21 -+ 28)12 (lI->-23)

-15 (40+25)O (21 -»• 2 l )

Proportionalchanges (%)

406070

35n o

- 3 50

Table 1. Changes in the concentrations of major constituents of blood plasma, also ofdry weights of tissues in Chinese mudskippers desiccated slowly until they lost 20 % ofinitial body weight*

Concentration

Blood plasmaOsmotic concentration (m-osmol/1)Na+ (m-equiv/1)Cl- (m-equiv/1)

Tissue dry weights (all %)White muscleHeartLiverBrain

• See Fig. 3-5 for data.t Initial values are means for baseline fish (o % weight loss). Values at 20 % weight loss are estimated

from least squares regression lines for plasma concentration or tissue dry weight w. body weight loss.

(repeatedly and clearly observed in fish kept in the outdoor tank at the laboratory).This is lost when the fish tries to catch a food item on the mudflat. This water isreplaced at the next water contact.

Fish out of water at night are normally quiescent on the mudflat unless disturbedby an intruder. They apparently may remain out of contact with water under thesequiet, dark, cool conditions for extended periods, perhaps up to several hours.

At least in early autumn, when environmental conditions during mid-day periodsare somewhat less extreme than they are in mid-summer, P. cantonensts shows nosignificant changes in its behaviour with respect to contact with water in response towide variations in environmental heat loadings and evaporative water loss rates.

Rates of evaporative water loss, and body temperatures. Rates of evaporative waterloss, estimated as changes in body weight, varied with the severity of applied desic-cation stresses (Fig. 2). Fish in still air and shade lost weight at about 6% h"1,fish in moving air and shade lost weight at rates up to about 20% h"1, and fish inmoving air and full sun lost weight at rates of about 45 % h-1. Tolerance for mag-nitude of dehydration was inversely related to rate. Fish in still air and shade surviveduntil total weight losses averaged 22 %; fish in moving air and shade tolerated weightlosses averaging 14%; and fish in moving air and sun tolerated weight lossesaveraging 8%.

Body temperatures for fish dehydrated under shade conditions were always belowambient air temperatures and approached wet bulb thermometer temperatures infish exposed to moving air. Fish exposed to moving air and bright sun either main-tained body temperatures at the level of shade air temperatures, or heated up 1-2 °Cby the time of death.

Frogs {Rana tigerina) subjected to desiccation under conditions of still air andshade lost weight at an average rate of 8% h-1. This rate was one-third higher thanthat for comparably treated mudskippers, despite the fact that the frogs averaged10-30 times the body weights of the fish, (28-34 % vs- i"i-3"2 g) and therefore hadsubstantially smaller surface/volume ratios.

Terrestrial life in amphibious fishes. Ill

co

g

300

200

io

• o

100 Jr

800

600 I

400 5eo

10 20Body wt loss (%)

Fig. 3. Changes in the concentrations of three major constituents (osmolality, Na+, Cl~) inthe blood plasma of Chinese mudskippers subjected to both rapid and alow dehydration.Points at zero weight loss are means ± 1 8.B. for groups of six control fish. All other pointsrepresent one fish each. Osmolality: x (slow dehydration only); Na+: A (fast dehydration),O (slow dehydration); Cl~: A (fast dehydration), • (slow dehydration).

Distribution of water and some solutes in the bodies of fish subjected to desiccation.Dehydration losses of body water appear to derive to different degrees from differenttissues. Some internal redistributing of osmotically active solutes also may occur.Of the tissues studied (blood, white muscle, heart, liver, brain), during slow desic-cations the heart sustained the largest proportional water loss, the blood and whitemuscle intermediate losses, and the brain and liver (which actually increased inwater content) the smallest losses (Fig. 3-5, Table 1). Speed of dehydration had

significant effects upon the identity of tissue water sources.Plasma osmolality increased twice as rapidly, in slow desiccation, as would be

66 M. S. GORDON, W. W.-S. NG AND A. Y.-w. YIP

50 -

40

30

20

10

0

oo

o

o Ao QO

AA

A AA

0 10 20 30Body wt loss (%)

Fig. 4. As Fig. 3, but for changes in dry weights of trunk white muscles (A, fast dehydration;O» slow dehydration) and heart (A, fast dehydration; • , slow dehydration).

expected if total body water losses were uniformly distributed among all tissues(Table 1). Plasma Na+ and Cl~ concentrations increased half again as rapidly. Theslopes of the relationships for the ionic changes were the same for both fast and slowdehydration (Fig. 3). This lack of proportionality between changes in osmolalityand major ionic concentrations may imply significant changes in concentrations ofother plasma solutes (see data on nitrogen metabolism below). However, the absolutincrease in osmolality nearly equals the sum of the Na+ and Cl~ increases.

Terrestrial life in amphibious fishes. HI 67

50

40

30

1

20

10

AA

AA

o

0 10 20 30Body wt loss (%)

Fig. 5. As Fig. 3, but for changes in dry weights of liver (A, fast dehydration;O, slow dehydration) and brain (•, slow dehydration only).

measurements of circulatory system and tissue extracellular space volumes are neededto determine the degrees to which plasma osmolality changes are due to water removaland/or solute shifts.

White muscle dry weight was unaffected by rapid desiccation up to 10% bodyweight loss (Fig. 4). Larger scale slow desiccation produced muscle dry weightchanges comparable in magnitude to those of plasma osmolality (Table 1).In contrast to this, heart dry weight increased proportionally 3 times faster,with the slopes of changes produced by both rapid and slow desiccation beingidentical.

Liver dry weight was unaffected by rapid desiccation up to 10% body weight

68 M. S. GORDON, W. W.-S . NG AND A. Y.-w. YIP

250

200

o1. 15°

100

50

00 10 20 30

Temperature (°C)Fig 6. Weight-specific rates of oxygen consumption for small to medium size Chinese mud-skippers in air equilibrated sea water and in air for 3-5 h, at three temperatures (io°, ao° and30 °C). Six fish in each group. Plotted points are means; vertical lines ± 1 S.E. Fish in seawater, • ; fish in air, O-

Terrestrial life in amphibious fishes. HI 69

Table 2. Ammonia and urea excretion by Chinese mudskippersin sea water at 19-21 °C

[All figures mean ± i S.E. (N).]

Excretion rates, * \ Urea-N as pro-

NH, —N Urea-N portion of total(mM N kg"1 h"1) (mM N kg"' h"1) N excretion (%)

Fed in previous 24 h 6'4±o-7(7) i»'6±a'o(7) 66±6Starved for 9-5 days 63 ± 0 3 (6) 74±i°(6) 55 ±3

loss (Fig. 5). Larger scale slow desiccation produced substantial decreases in dryweight. Brain dry weight was unaffected by large scale slow desiccation.

Metabolic rates. The weight-specific resting (very near standard) rates of oxygenconsumption of small to medium-size fish at each of three test temperatures (io°, 200

30 °C) were equal, independent of body weight, over the size range o!i-i*5 g. Theserates also were constant over long periods of time (up to 6-5 h) during which thefish were confined to small microrespirometer chambers.

The presence or absence of sea water in the respirometer chambers also had nostatistically significant effect on metabolic rates of these fish at any of the three testtemperatures (Fig. 6). Metabolic sensitivity to temperature was moderate between10° and 20 °C (Q10 ^ 1-3 for fish in sea water, 1-7 for fish in air), but was fairlyhigh between 20° and 30 °C (Q10 ~ 2-7 for fish in sea water, 2-5 for fish in air).

Routine metabolic rates for medium sized and somewhat larger fish in sea waterwere measured only at 20 °C. As usual with measurements of routine rates, the resultswere variable (160-430 ml Oa kg"1 h"1). This range of variability was constant overthe body weight range o-8-4>7 g. The maximum routine rate measured was 5 timeslarger than the resting metabolic rate for somewhat smaller fish at the same tem-perature.

Heart beat frequencies. Stabilized rates in air ranged from 96 to 125 min-1. Ratechanges following subsequent immersions or emersions were all within + 20% ofthese initial levels in individual fish. Directions of changes were variable. Some fishshowed no changes at all, some increased rate on immersion and decreased again onemersion, and some decreased rate on immersion and increased again on emersion.The average performance for the entire group was close to zero change in rate.

Lactic acid concentrations in blood. Blood lactate analyses were carried out onthree groups of six fish each, all at temperatures of 20—24 °C. Results were (mean ± S.E.) :Controls (fish allowed to move in and out of sea water undisturbed, ad lib), 14 + 2mg% (mg/100 ml blood). Fish in aerated sea water, no access to air for 20 h, 12 ± 2mg%. Fish in moist chambers, out of contact with liquid water for 20 h, 15 + 2 mg%.Thus, there were no statistically significant changes in blood lactate concentrationsas a result of fish being in or out of water for extended periods.

Ammonia and urea concentrations in blood. The fish were too small for us to deter-mine blood ammonia levels with the method available. However, based on work withother amphibious fish species (see Materials and Methods), blood ammonia probablyaccounted for about 3% of the total (ammonia + urea)-N which was measured.

Total (ammonia + urea)-N was determined in the blood of three groups of six

70 M. S. GORDON, W. W.-S . NG AND A. Y.-w. YIP

5 10Time (h)

Fig. 7. Weight-specific rates of excretion of waste urea-N and ammonia-N by medium-sixedChinese mudskippera continuously and fully immersed in sea water (horizontal lines labelled' control'), and immersed in sea water for indicated lengths of time after having been in air,in moist chambers, for 16-5 h. All measurements on groups of 6-7 fish starved for 9-5 days.Experimental temperatures 19-33 °C. Plotted points are means; vertical lines ± 1 S.E. Urea-Nexcretion rates: A, ; ammonia-N excretion rates: O, .

fish each, all at temperatures of 19-23 °C. Results were (mean ± S.E.): Fish fed withinprevious 24 h, in and out of sea water ad lib. 97 ± 6 mg I"1 (~ 3-4 mM urea). Fishstarved for preceding 9-5 days, in and out of sea water ad lib., 105 + 14 mg I-1 (^ 3-6mM urea). Fish starved for preceding 13-5 days, out of water in moist chambersfor 19 h, 240 ± 25 mg I"1 (~ 8-5 mM urea).

Ammonia and urea excretion rates. Both absolute rates and relative proportionsof ammonia and urea excretion were affected by both the nutritional status of fishand their having been out of contact with liquid water for extended periods oftime (Fig. 7, Table 2). Recently fed fish excreted twice as much urea-N than they didammonia-N.

Fish starved for 9-5 days excreted only 20% more urea-N than they did ammonia-N. Absolute rates of ammonia-N excretion were unaffected by starvation, but urea-Nexcretion decreased by 40%.

Fish starved for 9-5 days, kept in air in moist chambers for 16-5 h, then replacedin sea water, excreted both ammonia and urea at rates above control values for anumber of hours (Fig. 7). Urea-N excretion rates for about 4 h after return to waterwere 3 times control rates, ammonia-N excretion rates for about 5 h after return towater were 2 times control rates. These higher rates gradually declined back tocontrol levels after these time intervals.

The probable cause of this pattern was bodily retention of waste nitrogen producedduring the periods out of water, the release of accumulated wastes being added tothe underlying control rates of excretion following return to water.

Terrestrial life in amphibious fishes. HI 71

On the basis of this model, a calculation can be made of the average rates ofammonia-N and urea-N production by the fish while they were out of water. Theareas enclosed by the experimental and control curves in Fig. 7 indicate an averagerate of production of ammonia-N of 2-1 mM kg"1 h"1; for urea N the rate was 6-o mMkg"1 h"1. Comparison of these figures with control rates (Table 2) indicates that fishout of water produced waste nitrogen about 40 % less rapidly than did the same fishimmersed in sea water. Urea-N formed a substantially higher proportion (75%) ofthis reduced production than it did of control rates of production.

Dissolved oxygen concentrations in mudskipper burrows. The sandy mud of the man-grove forest flats inhabited by Hong Kong mudskippers is fine grained and dense(hence is not very porous or permeable), and is also highly organic (hence probablyhas high chemical and biological oxygen demands). Mudskippers often spend sig-nificant periods of time in their burrows in this mud (see Discussion). These burrowsmay be up to a metre or more in length and, at least in the flat we studied, werealways filled to their openings with water.

The dissolved oxygen concentrations in the water from 5 to 15 cm depth insidefive different burrows ranged from 0-2 to 0-7 ml I"1. Since the mud in the flats wasblack in colour from depths beneath the surface of less than 1 cm, these values prob-ably are high, and represent some admixture of better aerated surface water resultingfrom our sampling activities.

Tolerance for hypoxia. The Chinese mudskipper does not seem to be able to toleratethe normal oxygen concentrations occurring in its burrows. Seven fish allowed torespire down the oxygen concentrations in closed jars of sea water, all at temperaturesof 25-28 °C, became completely inert and inactive at dissolved oxygen concentrationsaveraging 0*8 ml I"1, ranging from 0-7 to i-o ml I"1. Thus, the minimum oxygenconcentration tolerated by any experimental fish was no lower than the maximumoxygen concentration measured at fairly short distances into the fishes' own burrows.

DISCUSSION

Like its East African sibling species Periophthalmus sobrinus (Gordon et al. 1969),P. cantonensis is a highly terrestrial fish well adapted to its mode of life. It can surviveout of water, under moderate environmental conditions, for up to z\ days. It doesnot drown in aerated sea water. In nature it spends over 90 % of its time out of waterthough it makes frequent brief contacts with water. It can tolerate evaporative waterlosses of over 20% of body weight, and loses water by evaporation at rates wellbelow those of frogs of much larger size treated similarly. It shows none of the majorfeatures of the 'diving syndrome', metabolic rates, heart rates, and blood lacticacid levels all being unaffected by shifts between water and air. It also becomes moreureotelic while in air. The overall patterns of physiological adjustments to life out ofwater are quite similar in both species.

We do not think it necessary to discuss these observations extensively here, sincemost of the relevant literature has been reviewed (see Gordon et al. (1969); Gordon(1970); Gordon et al. (1970); Graham (1973, 1976); Tamura et al. (1976)), but wewould like to make several additional comments.

First, the Chinese mudskipper definitely carries small volumes of water about with

72 M. S. GORDON, W. W.-S . NG AND A. Y.-w. YIP

it, in its buccal cavity, during excursions on dry land. The loss of this water (evidencedby two wet spots appearing on the mud surface directly below the opercular openings),usually as a result of an attempt to feed, seems to be one of the main stimuli promp-ting the fish to make contact with liquid water for brief periods at relatively shortintervals. This water presumably assists the fish in maintaining respiratory gasexchanges across its gills and bucco-pharyngeal membranes (Singh & Munshi, 1969).It presumably also assists in maintaining body hydration. There has been some diver-sity of opinion in the literature regarding the occurrence of this phenomenon (Gordonet al. 1968; Graham, 1976). An unanswered question is whether or not this watermay play a role in waste nitrogen excretion by fish out of water. Our experimentson waste nitrogen excretion did not permit fish out of water to retain or renew liquidwater in their buccal cavities.

Second, the present results indicate that phylogenetic diversity in physiologicaladaptations to terrestrial life on the part of amphibious fishes, although very large(Gordon et al. 1969; Gordon et al. 1970; Graham, 1973, 1976; Tamura et al. 1976)does not quite extend to the point where every species is substantially different fromevery other species. The present work is the first opportunity we have to compare indetail the performances in these respects of two phylogenetically closely related, butstill clearly systematically distinguishable, amphibious species. Evolutionary physio-logical differentiation in the P. koelreuteri group of mudskippers appears not tohave progressed very far, despite great geographical separation of the two speciesstudied (east Africa and east Asia).

Third, one of the respects in which some small scale physiological differentiationmay have occurred relates to waste nitrogen excretion. P. sobrinus increased itsrates of ammonia and urea production above control levels while it was out of water(Gordon et al. 1969; Gordon, 1970). P. cantonensis, however, decreased these rates.

Fourth, the shifts toward ureotelism which occur during periods out of water in allthree of the amphibious fish species studied in this respect to date [P. cantonensis(this paper); P. sobrinus (Gordon et al. 1969); Sicyases sanguineus (Gordon et al.1970)] may be relevant in discussions of the possible evolutionary adaptive value ofureotelism in the lower vertebrates. Thomson (1971) states that ureotelism increasesin lungfishes whenever they lower ventilation rates of their gills and increase aerialrespiration (causes of such shifts may be either the initiation of aestivation, or exposureto low aquatic oxygen tensions or high aquatic carbon dioxide tensions). He suggeststhat this shift in nitrogen metabolism results in avoidance of possible ammoniatoxaemia, since most ammonia excretion by fishes occurs by way of the gills. Thisanalysis could also plausibly explain the occurrence of the shifts toward ureotelismin the three amphibious fish species. If this is so, this mechanism is phylogeneticallywidely distributed among living fishes and may have been one of the pre-existingphysiological properties which played an important role in the evolution of terres-triality. Janssens (1972) demonstrated the occurrence of exactly this process in theaquatic anuran amphibian Xenopus laevis.

Fifth, it is possible to test, by means of a calculation, the plausibility of the idea thatmudskippers accumulate in their bodies the waste nitrogen they produce duringperiods out of water. Starved fish out of water for 19 h had blood urea-N levelsnear 17 mia 1-1. Starved fish in water had blood urea-N levels near 7 mM I"1. Nor-

Terrestrial life in amphibious fishes. Ill 73

mally hydrated mudskippers have 76% water in their bodies (Gordon, unpublishedobservations). Assuming uniform distribution of urea in all body water, completebodily retention of all urea-N produced while the fish were out of water, and aconstant rate of urea production during the time out of water, 2 g weight fish accum-ulated in their bodies 13 fiM of urea-N during 16-5 h periods out of water (see Fig. 7).This amount may be compared with a total of 200 /JM of urea-N in excess of controlexcreted by the experimental fish ((6 /IM g"1 h"1) (16-5 h) (2 g)). The large disparitybetween the two figures supports the idea of waste nitrogen retention, and bringsup further questions for study. What are both the locations and the chemical identitiesof the molecules accumulating in these fish which result in the release of so muchurea after they return to water?

Three additional topics remain: the internal sources of water lost by the fish duringepisodes of desiccation; the dependence of oxygen consumption rates on temperatureand activity; and the tolerance of mudskippers for hypoxia.

The pattern of desiccational water loss from the tissues of Chinese mudskippersis different in detail from the patterns occurring in anuran amphibians (Adolph,1933, 1943; Hillman, 1977; Shoemaker, 1964; Smith & Jackson, 1931). The mudskip-per pattern also differs somewhat from the pattern of osmotic water losses from thetissues of the marine teleost black-chin surf perch (Waggoner, 1972). The mostimportant possible uniformity in all these varied patterns is that these differentanimals appear to maintain central nervous system hydration as much as possible.

The measured metabolic responses of Chinese mudskippers to temperature fitin well with their natural behaviour patterns. This is particularly so with respect totheir low metabolic rates at low temperatures. Hong Kong, although tropical forabout 8 months of each year, is temperate during the remaining 4 months. DuringJanuary and February of each year sea level air temperatures regularly go down to3-4 °C for periods of a few days to a few weeks. At these times the mudskippersdisappear from the surfaces of their mudflats and retire to their burrows. If one digsthem out, they are found to be still capable of moving, but are lethargic. Thus, theirburrows are important refuges during cold periods. Kobayashi, Dotsu & Takita(1971) have made similar observations in southern Japan. We do not know if mud-skippers are capable of metabolic acclimation to temperature.

These observations, together with the patterns of burrow use occurring duringwarmer periods, bring up an additional question. Are mudskippers capable of meta-bolizing anaerobically for periods like those they appear to spend in their burrows?Our oxygen tolerance results make this seem unlikely. Direct measurements of lacticacid production in hypoxically stressed Australian mudskippers {Periophthalmusvulgaris, another member of the P. koelreuteri group) support this view (Gordon,in preparation). Further work is needed to determine whether mudskippers overcomethe problems of oxygen shortage in their burrows by behavioural or physiologicalmeans.

Our observations of routine metabolic rates indicate that mudskippers at moderatetemperatures have sufficient metabolic scope to support the short bursts of high levelactivity which they perform when they skip rapidly away from threats of variouskinds. Additional work on Australian mudskippers, however, indicates that thesefish do not have much staying power at high activity levels (Gordon, in preparation).

74 M. S. GORDON, W. W.-S. NG AND A. Y.-w. YIP

Our measurements of rates of oxygen consumption differ in several respects fromthose reported by Tamura et al. (1976). The shapes of the metabolic rate vs. tempera-ture curves for fish in air and in water are nearly identical in our results (both expon-ential in form), but are substantially different in theirs (water curve nearly linear,air curve exponential). The two sets of values differ at several points. The onlytemperature interval in which they found no statistically significant differencesbetween rates in air and in water was io°-i5 °C. We found no differences over theentire range ioo~3O °C.

The reasons for these differences are not clear. Several plausible possibilities are:(a) Seasonal effects (our measurements were made in November and December;Tamura et al. do not say at what time of year they did their work, but the contextof the paper makes summer seem probable); (b) micro-evolutionary differencesbetween geographically isolated populations (Gordon et al. (1968) discuss differencesin behaviour between populations of Periophthalmus sobrinus in Madagascar andMozambique); and (c) differences in states of thermal acclimation of the two groupsof fishes (our measurements were made within a few hours of the fish having beenplaced at test temperatures; theirs were made after a full day's acclimation at eachtest temperature). Possible adaptive values for the differences cannot be determinedfrom the information at hand.

These studies were carried out at the Marine Science Laboratory, Chinese Univer-sity of Hong Kong (CUHK), Shatin, New Territories, Hong Kong. The work wassupported by CUHK; by the Zoology-Fisheries programme of the University ofCalifornia, Los Angeles; by U.S. National Science Foundation research grantGB 31180 to the senior author; and by a Senior Queen's Fellowship in MarineScience of the Australian Government, also to the senior author. We wish to thankthe following people for their kind assistance and co-operation. Dr Lamarr Trott,then Director of the Marine Science Laboratory; Dr. Li Choh-ming, Vice-Chancellorof CUHK; the technical staff of the Marine Science Laboratory, CUHK; and Profes-sor G. A. Horridge, Department of Neurobiology, Australian National University,Canberra, in whose Department much of this paper was written. S. Hillman madeseveral useful suggestions.

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