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Osmotic and ionic balance in two species of Cenocorixa (Hemiptera)
J. Insect Physiol., 1972, Vol. 18, pp. 883 to 895. Pergamon Press. Printed in Great Britain
OSMOTIC
AND IONIC
BALANCE IN TWO SPECIES CENOCORIXA (HEMIPTERA)
G. G. E. SCUDDER,
M. S. JARIAL,*
OF
and S. K. CHOYt
Department of Zoology, University of British Columbia, Vancouver 8, B.C. (Received 17 March 1971; revised 2 November 1971) Abstract-Two species of Cenocorixa occur in central British Columbia, C. bij%a in waters with conductivities from 27 to 20,000~-mhos/cm and C. expleta in waters with conductivities between 5000 and 30,000~-mhos/cm. The osmotic and ionic balances of the two species in various concentrations of a single lake water are similar: the response is that of a typical fresh-water insect. Neither species is able to hyporegulate in the higher salinities and neither is able to produce hyperosmotic urine. Marked mortality occurs when the haemolymph osmotic response curve crosses the isosmotic line: this is at AI 0-90X for C. bifida and AE l-22’% for C. e&eta. A difference in their physiological tolerances seems to be the factor determining the differential distribution of the two species in saline waters. C. expletu can survive in low salinities, although it does not naturally occur in fresh water. Ecological interactions with C. bijda would seem to be important in excluding C. expleta from fresh-water habitats. INTRODUCTION
WHILE much is known about the general ecology of Corixidae, little work has been done on their physiology. Although there are a number of species that occur in saline water, to date only CLAUS (1937) has studied and compared the salinity tolerance and osmotic balance of various fresh- and brackish-water species. The distribution of Corixidae in a series of inland saline lakes in central British Columbia has been described by SCUDDER(1969a, b), and two species of Cerzocorixa are recorded in this area. SCUDDER(1969a) notes that while one species, Cenocorixa b$idu (Hung.) occurs in waters whose conductivities range from 27 to 20,000 p-mhos/cm, another species, C. expleta (Uhler) occurs naturally in waters that have conductivities between 5000 and 30,000 El-mhos/cm. Further, SCUDDER(1969b) sh ows by experiments in which temperature and salinity are varied that C. bijda cannot survive in water whose conductivity is above 20,000 pmhos, but C. expleta evidently can, C. expleta was also observed to be able to live for some time in the lower salinities. This paper reports on a comparative study of the osmotic and ionic balances in these two species of Corixid. It is an attempt to understand further the distribution pattern of these two species. * Present address: Department of Anatomy, College of Osteopathic Medicine and Surgery, Des Moines, Iowa. t Present address: Department of Fisheries, Kuala Trengganu, Trengganu, Malaysia. 883
884
G. G. E.
SCUDDER, M. S. JARIAL,ANDS. K. CHOY
MATERIALS AND METHODS Adult insects were taken in the fall from natural populations and transported to the laboratory in 1 gal Thermos jugs half filled with lake water. Some weeds were available for the insects to cling to. C. expleta adults were taken from a lake (LB2), located near Lac du Bois in the hills north of Kamloops, B.C. and C. bifida was collected from White Lake, on the Green Timbers Pleateau near Clinton, B.C. and from Lake Lye ( = Box 20-21 in SCUDDER,1969a, b), on Beecher’s Prairie in the Chilcotin region of British Columbia. In the laboratory, insects were held temporarily in 30 x 24 x 10 cm covered Plexiglas dishes which contained natural lake water and weeds. These dishes were kept in a constant temperature cabinet maintained at 5°C and 6 hr light : 18 hr dark photoperiod. Insects were used for experiments within 1 week of capture. Water from Long Lake on the Green Timbers Plateau was used in all the experiments. Its ionic composition is given in Table 1. Different concentrations were prepared by dilution with glass-distilled water or by evaporation at room temperature: all waters were passed through a Millipore HA (0.45 p) filter. TABLE~-COMPOSITION0~ LONGLAEEWATER (MAJOR IONS)USED TOPRODUCE VmIous MEDIA FOREXPERIMENTS Ion Na+ K+ Ca2+ Mg2+
CO,%HCO,Clso,2-
m-equiv/l.
Total cations (%I
158.0 9.8 0.5 12-6
87.3 5-4 0.3 7.0
31.8 34.3 21.7 96.1
Total anions (%f 17.0 19.2 11.8 52.0
In the experiments using these waters, 150 ml of water was put into a covered 250 ml beaker, and to this was added insects and a 4 x 6 cm piece of plastic screen temperature for the insects to cling to. The beakers were kept in a unsent cabinet under the conditions noted above. During the experiments, the insects were not fed, since they had an adequate reserve of fat body, being collected when about to enter the overwintering period: such unfed insects could be kept alive for some 12 weeks. For collection of haemolymph samples, insects were surface-dried on Kleenex tissue, held in a piece of Parafilm M, and the right forewings were removed with forceps. The haemolymph which issued from this wound was collected in a 1~1 disposable glass micro-pipette (Microcap) (Drummond Scientific Co., Broomall,
OSMOTIC
AND IONIC BALANCE
IN TWO
SPECIFSOF CENOCORIXA
88.5
Pa.). Urine was collected from surface-dried insects held on Parafilm M, by gently squeezing the abdomen and collecting the clear fluid that issued from the anus. Fluid from the ileum was obtained by dissecting the insect in insect Ringer and ligating this region of the hind-gut fore and aft with sira wax drawn into threads. The ligated portion of the gut was then removed, placed under liquid paraffin, and the contents collected in a micro-pipette as they issued from a small incision made between the ligatures. In all samples, measurements were made on individual insects: pooled samples were used only in the determination of the concentrations of sodium in the haemoSamples for determination of freezinglymph during the acclimation experiments. point depression were collected and O-1 ~1 portions were sandwiched between liquid paraffin in 1~1 Microcaps. The melting point was recorded after freezing with dry ice. Samples taken for determination of ions were 1~1 volumes of haemolymph. All samples were studied immediately after collection, since freezing can bring about pronounced changes in the haemolymph (STEPHEN and JOHNSON, 1962). Freezing-point depressions of experimental medium, ileum content, haemolymph, and urine were determined by the microcryoscopic method of RAMSAY and BROWN (1955). Chloride in lake water was measured by use of a Cotlove chloridometer, while chloride in haemolymph and urine were determined by potentiometric titration using a Radiometer (model 25SE) pH meter after the method of RAMSAY et al. (1955). Potassium and sodium in lake water was measured by use of a Zeiss PF5 flame photometer, the potassium values being determined with a sodium and calcium chloride swamp solution so as to provide a constant background. Sodium and potassium in the haemolymph were estimated on a Unicam SP900 flame spectrophotometer, the samples for potassium analysis being diluted in a sodium chloride swamping solution. In the determination of body water, the insects were removed from the water, surface dried with Kleenex tissue, lightly etherized, and placed in small preweighed aluminium foil dishes. These dishes were then put in an oven at 98°C for 1 min to remove additional surface moisture and then weighed: extraneous water caused great errors in determinations if not removed. After obtaining the initial wet weight, the insects were then dried at 98°C to a constant weight (72 hr). In the final determination of the dry weight, the dish containing the insect was allowed to cool for 1 hr at 18°C and 22% r.h. All weighing was done on a Mettler Grammatic balance, accurate to 0.001 g. The percentage of body water was calculated and expressed in terms of the insect wet weight. In the preliminary analysis of results, data from male and female, as well as from flying and non-flying morphs, were gathered and compared. Since no significant difference was found in these data, no reference to sex or morph is made in the results presented. All values are based on data from at least 10 insects. In the results, a clear distinction is made between data obtained from animals in experiments where there was little or no mortality, and data from experiments
886
G. G. E. SCUDDER, M. S. JARIAL, ANDS. K. CHOY
in which the mortality was high (see SCUDDED, 1969b for mortality data). In the graphs, a broken line is used through points obtained in experiments with high mortality. Data from such experiments cannot be regarded as truly indicative of the physiological attributes and capacity of the species as a whole, and may not represent a steady state (equilibrium) condition. RESULTS
Acdimatiim
Acclimation was studied by placing insects in media either more concentrated or more dilute than the natural lake water in which they had been kept. Freezingpoint determinations and sodium values for the haemolymph were determined and both species showed a similar response to change (Figs. 1, 2). Considerable
4
i
Time (hours)
FIG. 1. Acclimation of haemolymph osmotic pressure in two species of Cenocor~xu. 0, C. bifida from A, 0*2S”C to A, O-61’% ; 0, C. bzjidu from A, O-25°C to AB 0.06°C; cf, C. ezpleta from A, 0*61”C to A, O-25°C.
alteration occurred in the first 24 hr, but statistically there were no significant differences between values obtained at 48, 72, and 96 hr : 72 hr was considered to be an adequate period for acclimation and all subsequent measurements were _~ made on insects acclimated for this period. Generat water balance A study of the water content of the body in the two species over the range of salinities selected shows that there is considerable regulation of the body water
OSMOTIC
AND
IONIC
BALANCE
IN TWO
SPECIES
OF
887
CENOCORIXA
100
sot 0
,
,
I
I
I
I
6
12
24
48
72
96
Time (hours)
FIG. 2. Acclimation of haemolymph sodium in two species of Cenocorixu. 0, C. bij%a from 47 m-equiv/l. to 212 m-equiv/l. ; 0, C. bifida from 47 m-equiv/l. to 25 m-equiv./l.; 0, C. expleta from 212 m-equiv/l. to 47 m-equiv/l.
and that the two species are not significantly different over most of the range. C. expletu showing a departure only in the highest salinity tested for this species (Fig. 3). Haemolymph osmotic balance
Freezing-point depressions for haemolymphs of insects placed in different media are shown in Fig : 4. C. bijida and C. expbta hyperregulate over the range of AE 0.05 to 0.5”C and there is no significant difference between the two species when insects are kept at these concentrations for 72 hr. Between AE O-56 to 1~26°C the species differ significantly. C. bi$da becomes isosmotic about A, 0.9O”C. C. expZetu, on the other hand, tends to conform over the range AE 05 to 1*22”C, although the haemolymph is slightly hyperosmotic to the medium: C. expZeta becomes isosmotic with the medium around AE 1.22”C. In the higher salinities, where there is marked mortality, survivors at the end of 72 hr have haemolymph hyposmotic to the medium. Urine osmotic balance
Determination of the freezing-point depressions of urine samples show that, over the range of salinities studied, neither species is able to produce urine hyperosmotic to the haemolymph (Fig. 5): the response cures of the two species are virtually identical. In salinities below An 0*4”C, the urine is hyperosmotic to the medium, whereas above AE 0*45”C it is slightly hyposmotic (Fig. 6). Both species
G. G.
30
E. SCUDDER, M. S. JARIAL, AND S. K. CWOY
; 0.0
I 0.2
I 0.4
I 0.6
I 0.0
Freezing point depression
1 1.0
of medium
, I.2
1 1.4
(“C)
FIG. 3. Water content of two species of Cenocorixa after 72 hr in various concentrations of Long Lake water. 0, C. bifida; l , C. expteta. 1.4 8 t..
rp d % f
75
1.0
% 5 8
J
E
on
m .I:
0.6
2 i 0.0 0.0
0.2
0.4
0.6 Freezing
pofnt
0.8 depression
1.0 of medium
1.2
1.4
1.6
(“C)
FIG. 4. Freezing-point depression of haemolymph of two species of Cemcorixa after 72 hr in various concentrations of Long Lake water. 0, C. ~~a; I), C. expleta.
produce copious urine in the low salinities, but very little urine is obtainable from insects in high salinity waters. Iimic &dame
of the ~~~~~~~
Data on haemolymph ion values in the two species are presented in Figs. 7 to 9. The sodium response curves for both species show a hyperregulation over the range 0 to 150 m-equiv/l. : no abrupt changes are observed in the curves, those
889
OSMOTIC AND IONIC BALANCE IN TWO SPECIES OF CENOCORIXA
D.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
/ 0.2
I
I
0.3
0.4
1 0.5
I
0.6
Freezing
I
I
07
0.8
point depression
r 0.9
of haemolymph
I
I
1.0
1.1
1
1.2
(“C)
FIG. 5. Relationship between freezing-point depression of urine and haemolymph in two species of Cenocorixa after 72 hr in various concentrations of Long Lake water. 0, C. bzj%iz; 0, C. e&eta.
0.0
0.2
0.4
0.6
Freezing point depression’of
0.8 medium
1.0
1.2
1.4
(‘C)
FIG. 6. Freezing-point depression of urine of two species of Cenocorixa after 72 hr in various concentrations of Long Lake water. 0, C. bifida; 0, C. expleta.
890
G. G. E. SCUDDER,M. S. JARIAL, AND S. K. CHOY 300 /
250 --I r %
____---~--~ zoo-
d .c I s P .z .s
100 -
ii
0
50
100
150
200
250
300
350
I 400
1 450
FIG. 7. Sodium balance in haemolymph of two species of Ceptocori~ after 72 hr in various concentrations of Long Lake water. 0, C. &&%a; l , C. expleti.
K+
in medium
(meq/l)
FIG. 8. Potassium balance in haemolymph of two species of Cenocorixa after 72 hr in various concentrations of Long Lake water. 0, C. b$k%z;l , C. ex#&z.
OSMOTICAND 1ONICBALANCEIN TWO SPECIES OF
CENOCOIUXA
891
for the two species being quite similar. However, there is a tendency for 42. ez$&z to h~ore~ate above this level, whereas C. ~~~ does not survive. The responses of the two species to the higher saiinities are therefore significantly different. 140
1
120 -
Cl-
in medium
(meqli)
FIG. 9. Chloride balance in baemol~ph
of two species of Cen~c~~ after 72 hr in various concentrations of Long Lake water. 0, C. bifida; 0, C. e~Ze~u.
Further, while C. expleta was able to maintain a constant chloride level in the haemolymph over the range of salinities tested, C. b$a!u was unable to do so. The responses of the two species to changes in the external concentration of potassium are probably similar and the reason for the irregularities in Fig. 8 in the response of C. exp&z is unknown: the experiment was repeated twice with essentially the same results. It should be noted that the range of salinities used did not involve high concentrations of chloride. The mortality found in the high salinities is no doubt due to other ions: the full regulatory powers of the two species with respect to chloride were thus probably not tested. DISCUSSION
Animals that are restricted to low salinities are unable to hyporegulate in highly saline waters, whereas the forms that are adapted to life in higher salinities can hyporegulate (BEADLE, 1969). The osmotic responses of C. bz$Wu and C. expZeta to the various concentrations of Long Lake water are similar, and typical of the responses of other fresh- or brackish-water insects (WI~~L~WOR~, 1938; BEADLE, 1943, 1969; SUTCLIFFE, 1961b). The response of C. b@&z is quite like
892
G. G. E. SCUDDER, M.
S. JARIAL,AND S. K. CHOY
that of the fresh-water Sigma ~~~~~~u Fieb (CLAUS, 1937). Further, the response of C. expletca, like the brackish water inhabiting S. ~~~~~~~ (Leach) ( = S. ~ugu~~~ Fieb.) studied by CLAUS (1937), shows that this species responds in a manner characteristic of a fresh-water form and not that of a salt-water form (BEADLE, 1939; CROGHAN,1958; NEMENZ, 1960; SUTCLIFFE, 1960; SHAW and STOBBART, 1963). That these two C~oc~ixa species are basically fresh-water insects is also suggested by the measurements of the urine osmotic pressures. At no time can these Corixidae produce a urine that is hyperosmotic to the haemolymph (Fig. lo), an ability that is regarded as an essential requirement of truly saline water forms (PHILXPs, 1970). A
lhmmlymph
lk”“,
C. bifida
&d”M
haemoiymph
lieurn
Rectum
c. expleta
FIG. 10. Freezing-point depression of haemolymph, ileum, and urine contents in two species of Cetzocorixa, under hydrated (0) and dehydrated (e) conditions. A, C. ~~~; B, C. ex@eta. The study of mortality for the two species in raised concentrations of Long Lake water (SCUDDER,1969b) clearly indicates an inability to survive in the saline environments. In waters where they are able to survive for considerable periods of time, the haemol~ph is hyperosmotic to the medium: in waters where they do not survive well, the few remaining insects had a haemolymph hyposmotic to the medium. However, these few insects did not survive much longer than 72 hr and the ability to hyporegulate is certainly not a characteristic on the species as a whole. A few surviving specimens might be in a transitory state of exchange and hence may not have reached a stage of equilibrium. Osmotic response curves, similar to those presented herein for these two Cenoco&~ species, have been described in the larvae of the Chironomids Chironomu~ phmosus (L.) and Procladius nubifer (Coq.) as well as the naiads of the damselfly Enallagma cZuu.wmMorse (LAIIER, 1969). LAUER(1969) concludes that
OSMOTIC ANDIONICBALANCE IN TwO SPECIES OFCENOCORIXA
893
these species can perform both hypo- and hyperosmotic regulation. However, the published data do not show if the hyporegulation is a property of the species as a whole, or of just a few individuals that survive a little longer than the rest. The haemolymph sodium, potassium, and chloride response curves in both species of Cenocorixa demonstrate an ability to maintain a rather constant level over a considerable range of external change, with C. expleta able to encompass slightly higher salinities than C. bi~da. However, where there is considerable mortality, this regulating ability does not persist. Instead, the levels of these three ions approach those in the medium, probably indicating a failure of the organs concerned with osmotic and ionic balance in the animals. The response curve for sodium, especially that of C. expleta, is more typical of an insect, such as the Trichopteran Ljmneph~t~ afinis Curtis (SUTCLIFFE,1961a) that is tolerant to saline conditions: it is not like that of typical fresh-water insects such as the Neuropteran SiaEis h&aria (L.) (SHAW, 1955) and the Trichoptera Anabolia neruosa Leach and Limnephilw stigma Curtis (SUTCLIFFE,1961b). However, over the limited range tested, the response curves for chloride and potassium, in both species of Cenocorixa, are clearly a response typical of fresh-water insects. We conclude that both C. ~~~ and C. e~p~ta are fresh-water insects, with an equal ability to regulate and survive in various fresh-water environments. They differ in their tolerance of high osmotic pressure and somewhat in their ability to regulate sodium, and hence to survive in the more saline waters. From the haemolymph osmotic data it seems that death occurs when the osmotic response curve crosses the isosmotic line. In C. bi$da this occurs at A, 0*9O”C, and in C. expletu at A, l+ZZ”C. These values correspond to water from Long Lake with concentrations of 20,500 and 28,000 p-mhos/cm, at 25°C respectively. These values are consistent with the field data which show that C. bz;fda occurs in waters with conductivities in the range of 27 to 20,000 p-mhos/cm (at 25°C) and C. expleta in waters up to 30,000 CL-mhoslcm (SCUDDER,1969a). Since the osmotic responses of haemolymph and urine, and the data on concentrations of sodium, potassium, and chloride in the haemolymph indicate that there is no significant difference between C. bi$da and C. expleta at the low salinities, one might conclude that both species are able to live equally well in fresh water. However, the field data show that while C. bi$da is present and breeds in fresh water C. expZeta does not (SCUDDER,1969a, b). The absence of the more saline tolerant C. expleta from freshwater is not correlated with an inability to regulate the internal milieu in this environment over a short period of time, and the species can certainly live in fresh water for a considerable period (SCUDDER, 1969b). C. expleta is like many other saline tolerant forms that are capable of hyperosmotic regulation in fresh water or solutions of low concentration (NEMENZ, 1960; POTTS and PARRY,1964; PHILLIPS, 1970). For example, an ability to survive and regulate in fresh water has been demonstrated for the saline tolerant Aedes cumpestris D. & K. (PHILLIPS and MEREDITH, 1970), though this species has only been taken in rather saline habitats and does not occur in numerous neighbouring fresh water localities (SCUDDER,1969b). Again, while Procladius nubifer lives in
894
G. G. E. SCUDDER, M. S. JARIAL,ANDS. K. CHOY
saline lakes, it can survive in fresh water, and yet is recorded to have disappeared from Lenore Lake in Washington when the T.D.S. dropped from 6-O to 2.3 g/l. (LAUER, 1969). It would seem that the absence of C. expleta from various fresh-water
lakes is not likely to be due to an inability to survive in these waters. Ecological factors such as predation, availability of suitable food, interspecific competition, and other ecological factors may be important (BEADLE, 1943). As noted by LAUER (1969), in these species that live in inland saline waters “the ecological interactions may be most decisive in determining distribution in water of lower salinity, with physiological tolerance limits of the species becoming the more important determining factor at higher salinity”. Acknowledgements-This research was supported by a grant to G. G. E. S. from the National Research Council of Canada. We wish to thank Dr. A. B. ACTONand Dr. J. E. PHILLIPSfor helpful suggestions and for reading the manuscript, and Miss J. MEREDITH for technical assistance. REFERENCES
BEADLEL. C. (1939) Regulation of the haemolymph in the saline water mosquito larva Aedes detritus Edw. g. exp. Biol. 16, 346-362.
BEADLE L. C. (1943) Osmotic regulation and the faunas of inland waters. BioZ. Rew. 18, 172-183.
BEADLE L. C. (1969) Osmotic regulation and the adaptation of freshwater animals to inland saline waters.
ve’erh.int. Verein. Limn. 17, 421-429. Untersuchungen zur C)kologie der Wasserwanzen, mit besonderer Beriicksichtigung der Brackwasserwanze Sigura Zugubris Fieb. Zool. Jb. (Physiol.) 58, 365-432. CROGHAN P. C. (1958) The osmotic and ionic regulation of Artemia salina (L.). 3. exp. Biol. 35, 219-233. LAUJXR G. J. (1969) Osmotic regulation of Tanypus nubifer, Chironomus plumosus, and Enallagma clausum in various concentrations of saline lake water. Physiol. Zoiil. 42, 381-387. NEMJWZ H. (1960) On the osmotic regulation of the larvae of Ephydra cinereu. J. Insect Physiol. 4, 38-44. PHILLIPS J. E. (1970) Apparent transport of water by insect excretory systems. Am. 2001. 10, 413-436.
CLAUSA. (1937) Vergleichend-physiologische
PHILLIPSJ. E. and MEREDITH J. (1969) Active sodium and chloride transport by anal papillae of a salt water mosquito larva (Aedes campestris). Nature, Lond. 222, 168-169. Porrs W. T. W. and PARRYG. (1964) Osmoticand Ionic Regulation in Animals. Pergamon, Oxford.
RAMSAYJ. A. and BROWNR. H. J. (1955) Simplified apparatus and procedure for freezingpoint determinations upon small volumes of fluid. r. scient. Instrum. 32, 372-37.5. RAMSAYJ. A., BROWNR. H. J., and CROGHAN P. C. (1955) Electrometric titration of chloride in small volumes. g. exp. Biol. 32, 822-829. SCUDDER G. G. E. (1969a) The distribution of two species of Cenocorixa in inland saline lakes of British Columbia. J. ent. Sot. B.C. 66, 32-41.
SCUDDER G. G. E. (1969b) The fauna of saline lakes on the Fraser Plateauin British Columbia. Verb. int. Verein. Limnol. 17, 430-439. SHAWJ. (19.55) Ionic regulation and water balance in the aquatic larva of Sialis Zutaria. J. exp. Biol. 32, 353-382.
OSMOTIC ANDIONICBALANCEIN TWOSPECIESOF CENOCORIXA
895
SHAW J. and STOBBARTR. H. (1963) Osmotic and ionic regulation in insects. Adv. Insect Physiol. 1,315-399. STEPHEN W. P. and JOHNSON0. W. (1962) Qualitative changes in insect blood proteins after freezing. r. Kansas ent. Sot. 35, 189-196. SUTCLIPPED. W. (1960) Osmotic regulation in the larvae of some euryhaline Diptera.
Nature, Land. 187, 331-332. SUTCLIPPED. W. (196fa) Studies on salt and water balance in caddis larvae (Trichoptera)-I. Osmotic and ionic regulation of body fluids in ~~~~~~ afinis Curtis. J. exp. Biol.
38, 501-519. SUTCLIFFED. W. (1961b) Studies on salt and water balance in caddis larvae (Triehoptera)II. Osmotic and ionic reguIation of body fluids in Linmphilus stigma Curtis and Anabolia nervosa Leach. J. exp. Biol. 38, 521-530. WIGGLB~WORTH V. B. (1938) The regulation of osmotic pressure and chloride concentration in the haemolymph of mosquito larvae. J. ex$. Biol. 15, 235-247.
OSMOTIC
AND IONIC
BALANCE IN TWO SPECIES CENOCORIXA (HEMIPTERA)
G. G. E. SCUDDER,
M. S. JARIAL,*
OF
and S. K. CHOYt
Department of Zoology, University of British Columbia, Vancouver 8, B.C. (Received 17 March 1971; revised 2 November 1971) Abstract-Two species of Cenocorixa occur in central British Columbia, C. bij%a in waters with conductivities from 27 to 20,000~-mhos/cm and C. expleta in waters with conductivities between 5000 and 30,000~-mhos/cm. The osmotic and ionic balances of the two species in various concentrations of a single lake water are similar: the response is that of a typical fresh-water insect. Neither species is able to hyporegulate in the higher salinities and neither is able to produce hyperosmotic urine. Marked mortality occurs when the haemolymph osmotic response curve crosses the isosmotic line: this is at AI 0-90X for C. bifida and AE l-22’% for C. e&eta. A difference in their physiological tolerances seems to be the factor determining the differential distribution of the two species in saline waters. C. expletu can survive in low salinities, although it does not naturally occur in fresh water. Ecological interactions with C. bijda would seem to be important in excluding C. expleta from fresh-water habitats. INTRODUCTION
WHILE much is known about the general ecology of Corixidae, little work has been done on their physiology. Although there are a number of species that occur in saline water, to date only CLAUS (1937) has studied and compared the salinity tolerance and osmotic balance of various fresh- and brackish-water species. The distribution of Corixidae in a series of inland saline lakes in central British Columbia has been described by SCUDDER(1969a, b), and two species of Cerzocorixa are recorded in this area. SCUDDER(1969a) notes that while one species, Cenocorixa b$idu (Hung.) occurs in waters whose conductivities range from 27 to 20,000 p-mhos/cm, another species, C. expleta (Uhler) occurs naturally in waters that have conductivities between 5000 and 30,000 El-mhos/cm. Further, SCUDDER(1969b) sh ows by experiments in which temperature and salinity are varied that C. bijda cannot survive in water whose conductivity is above 20,000 pmhos, but C. expleta evidently can, C. expleta was also observed to be able to live for some time in the lower salinities. This paper reports on a comparative study of the osmotic and ionic balances in these two species of Corixid. It is an attempt to understand further the distribution pattern of these two species. * Present address: Department of Anatomy, College of Osteopathic Medicine and Surgery, Des Moines, Iowa. t Present address: Department of Fisheries, Kuala Trengganu, Trengganu, Malaysia. 883
884
G. G. E.
SCUDDER, M. S. JARIAL,ANDS. K. CHOY
MATERIALS AND METHODS Adult insects were taken in the fall from natural populations and transported to the laboratory in 1 gal Thermos jugs half filled with lake water. Some weeds were available for the insects to cling to. C. expleta adults were taken from a lake (LB2), located near Lac du Bois in the hills north of Kamloops, B.C. and C. bifida was collected from White Lake, on the Green Timbers Pleateau near Clinton, B.C. and from Lake Lye ( = Box 20-21 in SCUDDER,1969a, b), on Beecher’s Prairie in the Chilcotin region of British Columbia. In the laboratory, insects were held temporarily in 30 x 24 x 10 cm covered Plexiglas dishes which contained natural lake water and weeds. These dishes were kept in a constant temperature cabinet maintained at 5°C and 6 hr light : 18 hr dark photoperiod. Insects were used for experiments within 1 week of capture. Water from Long Lake on the Green Timbers Plateau was used in all the experiments. Its ionic composition is given in Table 1. Different concentrations were prepared by dilution with glass-distilled water or by evaporation at room temperature: all waters were passed through a Millipore HA (0.45 p) filter. TABLE~-COMPOSITION0~ LONGLAEEWATER (MAJOR IONS)USED TOPRODUCE VmIous MEDIA FOREXPERIMENTS Ion Na+ K+ Ca2+ Mg2+
CO,%HCO,Clso,2-
m-equiv/l.
Total cations (%I
158.0 9.8 0.5 12-6
87.3 5-4 0.3 7.0
31.8 34.3 21.7 96.1
Total anions (%f 17.0 19.2 11.8 52.0
In the experiments using these waters, 150 ml of water was put into a covered 250 ml beaker, and to this was added insects and a 4 x 6 cm piece of plastic screen temperature for the insects to cling to. The beakers were kept in a unsent cabinet under the conditions noted above. During the experiments, the insects were not fed, since they had an adequate reserve of fat body, being collected when about to enter the overwintering period: such unfed insects could be kept alive for some 12 weeks. For collection of haemolymph samples, insects were surface-dried on Kleenex tissue, held in a piece of Parafilm M, and the right forewings were removed with forceps. The haemolymph which issued from this wound was collected in a 1~1 disposable glass micro-pipette (Microcap) (Drummond Scientific Co., Broomall,
OSMOTIC
AND IONIC BALANCE
IN TWO
SPECIFSOF CENOCORIXA
88.5
Pa.). Urine was collected from surface-dried insects held on Parafilm M, by gently squeezing the abdomen and collecting the clear fluid that issued from the anus. Fluid from the ileum was obtained by dissecting the insect in insect Ringer and ligating this region of the hind-gut fore and aft with sira wax drawn into threads. The ligated portion of the gut was then removed, placed under liquid paraffin, and the contents collected in a micro-pipette as they issued from a small incision made between the ligatures. In all samples, measurements were made on individual insects: pooled samples were used only in the determination of the concentrations of sodium in the haemoSamples for determination of freezinglymph during the acclimation experiments. point depression were collected and O-1 ~1 portions were sandwiched between liquid paraffin in 1~1 Microcaps. The melting point was recorded after freezing with dry ice. Samples taken for determination of ions were 1~1 volumes of haemolymph. All samples were studied immediately after collection, since freezing can bring about pronounced changes in the haemolymph (STEPHEN and JOHNSON, 1962). Freezing-point depressions of experimental medium, ileum content, haemolymph, and urine were determined by the microcryoscopic method of RAMSAY and BROWN (1955). Chloride in lake water was measured by use of a Cotlove chloridometer, while chloride in haemolymph and urine were determined by potentiometric titration using a Radiometer (model 25SE) pH meter after the method of RAMSAY et al. (1955). Potassium and sodium in lake water was measured by use of a Zeiss PF5 flame photometer, the potassium values being determined with a sodium and calcium chloride swamp solution so as to provide a constant background. Sodium and potassium in the haemolymph were estimated on a Unicam SP900 flame spectrophotometer, the samples for potassium analysis being diluted in a sodium chloride swamping solution. In the determination of body water, the insects were removed from the water, surface dried with Kleenex tissue, lightly etherized, and placed in small preweighed aluminium foil dishes. These dishes were then put in an oven at 98°C for 1 min to remove additional surface moisture and then weighed: extraneous water caused great errors in determinations if not removed. After obtaining the initial wet weight, the insects were then dried at 98°C to a constant weight (72 hr). In the final determination of the dry weight, the dish containing the insect was allowed to cool for 1 hr at 18°C and 22% r.h. All weighing was done on a Mettler Grammatic balance, accurate to 0.001 g. The percentage of body water was calculated and expressed in terms of the insect wet weight. In the preliminary analysis of results, data from male and female, as well as from flying and non-flying morphs, were gathered and compared. Since no significant difference was found in these data, no reference to sex or morph is made in the results presented. All values are based on data from at least 10 insects. In the results, a clear distinction is made between data obtained from animals in experiments where there was little or no mortality, and data from experiments
886
G. G. E. SCUDDER, M. S. JARIAL, ANDS. K. CHOY
in which the mortality was high (see SCUDDED, 1969b for mortality data). In the graphs, a broken line is used through points obtained in experiments with high mortality. Data from such experiments cannot be regarded as truly indicative of the physiological attributes and capacity of the species as a whole, and may not represent a steady state (equilibrium) condition. RESULTS
Acdimatiim
Acclimation was studied by placing insects in media either more concentrated or more dilute than the natural lake water in which they had been kept. Freezingpoint determinations and sodium values for the haemolymph were determined and both species showed a similar response to change (Figs. 1, 2). Considerable
4
i
Time (hours)
FIG. 1. Acclimation of haemolymph osmotic pressure in two species of Cenocor~xu. 0, C. bifida from A, 0*2S”C to A, O-61’% ; 0, C. bzjidu from A, O-25°C to AB 0.06°C; cf, C. ezpleta from A, 0*61”C to A, O-25°C.
alteration occurred in the first 24 hr, but statistically there were no significant differences between values obtained at 48, 72, and 96 hr : 72 hr was considered to be an adequate period for acclimation and all subsequent measurements were _~ made on insects acclimated for this period. Generat water balance A study of the water content of the body in the two species over the range of salinities selected shows that there is considerable regulation of the body water
OSMOTIC
AND
IONIC
BALANCE
IN TWO
SPECIES
OF
887
CENOCORIXA
100
sot 0
,
,
I
I
I
I
6
12
24
48
72
96
Time (hours)
FIG. 2. Acclimation of haemolymph sodium in two species of Cenocorixu. 0, C. bij%a from 47 m-equiv/l. to 212 m-equiv/l. ; 0, C. bifida from 47 m-equiv/l. to 25 m-equiv./l.; 0, C. expleta from 212 m-equiv/l. to 47 m-equiv/l.
and that the two species are not significantly different over most of the range. C. expletu showing a departure only in the highest salinity tested for this species (Fig. 3). Haemolymph osmotic balance
Freezing-point depressions for haemolymphs of insects placed in different media are shown in Fig : 4. C. bijida and C. expbta hyperregulate over the range of AE 0.05 to 0.5”C and there is no significant difference between the two species when insects are kept at these concentrations for 72 hr. Between AE O-56 to 1~26°C the species differ significantly. C. bi$da becomes isosmotic about A, 0.9O”C. C. expZetu, on the other hand, tends to conform over the range AE 05 to 1*22”C, although the haemolymph is slightly hyperosmotic to the medium: C. expZeta becomes isosmotic with the medium around AE 1.22”C. In the higher salinities, where there is marked mortality, survivors at the end of 72 hr have haemolymph hyposmotic to the medium. Urine osmotic balance
Determination of the freezing-point depressions of urine samples show that, over the range of salinities studied, neither species is able to produce urine hyperosmotic to the haemolymph (Fig. 5): the response cures of the two species are virtually identical. In salinities below An 0*4”C, the urine is hyperosmotic to the medium, whereas above AE 0*45”C it is slightly hyposmotic (Fig. 6). Both species
G. G.
30
E. SCUDDER, M. S. JARIAL, AND S. K. CWOY
; 0.0
I 0.2
I 0.4
I 0.6
I 0.0
Freezing point depression
1 1.0
of medium
, I.2
1 1.4
(“C)
FIG. 3. Water content of two species of Cenocorixa after 72 hr in various concentrations of Long Lake water. 0, C. bifida; l , C. expteta. 1.4 8 t..
rp d % f
75
1.0
% 5 8
J
E
on
m .I:
0.6
2 i 0.0 0.0
0.2
0.4
0.6 Freezing
pofnt
0.8 depression
1.0 of medium
1.2
1.4
1.6
(“C)
FIG. 4. Freezing-point depression of haemolymph of two species of Cemcorixa after 72 hr in various concentrations of Long Lake water. 0, C. ~~a; I), C. expleta.
produce copious urine in the low salinities, but very little urine is obtainable from insects in high salinity waters. Iimic &dame
of the ~~~~~~~
Data on haemolymph ion values in the two species are presented in Figs. 7 to 9. The sodium response curves for both species show a hyperregulation over the range 0 to 150 m-equiv/l. : no abrupt changes are observed in the curves, those
889
OSMOTIC AND IONIC BALANCE IN TWO SPECIES OF CENOCORIXA
D.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
/ 0.2
I
I
0.3
0.4
1 0.5
I
0.6
Freezing
I
I
07
0.8
point depression
r 0.9
of haemolymph
I
I
1.0
1.1
1
1.2
(“C)
FIG. 5. Relationship between freezing-point depression of urine and haemolymph in two species of Cenocorixa after 72 hr in various concentrations of Long Lake water. 0, C. bzj%iz; 0, C. e&eta.
0.0
0.2
0.4
0.6
Freezing point depression’of
0.8 medium
1.0
1.2
1.4
(‘C)
FIG. 6. Freezing-point depression of urine of two species of Cenocorixa after 72 hr in various concentrations of Long Lake water. 0, C. bifida; 0, C. expleta.
890
G. G. E. SCUDDER,M. S. JARIAL, AND S. K. CHOY 300 /
250 --I r %
____---~--~ zoo-
d .c I s P .z .s
100 -
ii
0
50
100
150
200
250
300
350
I 400
1 450
FIG. 7. Sodium balance in haemolymph of two species of Ceptocori~ after 72 hr in various concentrations of Long Lake water. 0, C. &&%a; l , C. expleti.
K+
in medium
(meq/l)
FIG. 8. Potassium balance in haemolymph of two species of Cenocorixa after 72 hr in various concentrations of Long Lake water. 0, C. b$k%z;l , C. ex#&z.
OSMOTICAND 1ONICBALANCEIN TWO SPECIES OF
CENOCOIUXA
891
for the two species being quite similar. However, there is a tendency for 42. ez$&z to h~ore~ate above this level, whereas C. ~~~ does not survive. The responses of the two species to the higher saiinities are therefore significantly different. 140
1
120 -
Cl-
in medium
(meqli)
FIG. 9. Chloride balance in baemol~ph
of two species of Cen~c~~ after 72 hr in various concentrations of Long Lake water. 0, C. bifida; 0, C. e~Ze~u.
Further, while C. expleta was able to maintain a constant chloride level in the haemolymph over the range of salinities tested, C. b$a!u was unable to do so. The responses of the two species to changes in the external concentration of potassium are probably similar and the reason for the irregularities in Fig. 8 in the response of C. exp&z is unknown: the experiment was repeated twice with essentially the same results. It should be noted that the range of salinities used did not involve high concentrations of chloride. The mortality found in the high salinities is no doubt due to other ions: the full regulatory powers of the two species with respect to chloride were thus probably not tested. DISCUSSION
Animals that are restricted to low salinities are unable to hyporegulate in highly saline waters, whereas the forms that are adapted to life in higher salinities can hyporegulate (BEADLE, 1969). The osmotic responses of C. bz$Wu and C. expZeta to the various concentrations of Long Lake water are similar, and typical of the responses of other fresh- or brackish-water insects (WI~~L~WOR~, 1938; BEADLE, 1943, 1969; SUTCLIFFE, 1961b). The response of C. b@&z is quite like
892
G. G. E. SCUDDER, M.
S. JARIAL,AND S. K. CHOY
that of the fresh-water Sigma ~~~~~~u Fieb (CLAUS, 1937). Further, the response of C. expletca, like the brackish water inhabiting S. ~~~~~~~ (Leach) ( = S. ~ugu~~~ Fieb.) studied by CLAUS (1937), shows that this species responds in a manner characteristic of a fresh-water form and not that of a salt-water form (BEADLE, 1939; CROGHAN,1958; NEMENZ, 1960; SUTCLIFFE, 1960; SHAW and STOBBART, 1963). That these two C~oc~ixa species are basically fresh-water insects is also suggested by the measurements of the urine osmotic pressures. At no time can these Corixidae produce a urine that is hyperosmotic to the haemolymph (Fig. lo), an ability that is regarded as an essential requirement of truly saline water forms (PHILXPs, 1970). A
lhmmlymph
lk”“,
C. bifida
&d”M
haemoiymph
lieurn
Rectum
c. expleta
FIG. 10. Freezing-point depression of haemolymph, ileum, and urine contents in two species of Cetzocorixa, under hydrated (0) and dehydrated (e) conditions. A, C. ~~~; B, C. ex@eta. The study of mortality for the two species in raised concentrations of Long Lake water (SCUDDER,1969b) clearly indicates an inability to survive in the saline environments. In waters where they are able to survive for considerable periods of time, the haemol~ph is hyperosmotic to the medium: in waters where they do not survive well, the few remaining insects had a haemolymph hyposmotic to the medium. However, these few insects did not survive much longer than 72 hr and the ability to hyporegulate is certainly not a characteristic on the species as a whole. A few surviving specimens might be in a transitory state of exchange and hence may not have reached a stage of equilibrium. Osmotic response curves, similar to those presented herein for these two Cenoco&~ species, have been described in the larvae of the Chironomids Chironomu~ phmosus (L.) and Procladius nubifer (Coq.) as well as the naiads of the damselfly Enallagma cZuu.wmMorse (LAIIER, 1969). LAUER(1969) concludes that
OSMOTIC ANDIONICBALANCE IN TwO SPECIES OFCENOCORIXA
893
these species can perform both hypo- and hyperosmotic regulation. However, the published data do not show if the hyporegulation is a property of the species as a whole, or of just a few individuals that survive a little longer than the rest. The haemolymph sodium, potassium, and chloride response curves in both species of Cenocorixa demonstrate an ability to maintain a rather constant level over a considerable range of external change, with C. expleta able to encompass slightly higher salinities than C. bi~da. However, where there is considerable mortality, this regulating ability does not persist. Instead, the levels of these three ions approach those in the medium, probably indicating a failure of the organs concerned with osmotic and ionic balance in the animals. The response curve for sodium, especially that of C. expleta, is more typical of an insect, such as the Trichopteran Ljmneph~t~ afinis Curtis (SUTCLIFFE,1961a) that is tolerant to saline conditions: it is not like that of typical fresh-water insects such as the Neuropteran SiaEis h&aria (L.) (SHAW, 1955) and the Trichoptera Anabolia neruosa Leach and Limnephilw stigma Curtis (SUTCLIFFE,1961b). However, over the limited range tested, the response curves for chloride and potassium, in both species of Cenocorixa, are clearly a response typical of fresh-water insects. We conclude that both C. ~~~ and C. e~p~ta are fresh-water insects, with an equal ability to regulate and survive in various fresh-water environments. They differ in their tolerance of high osmotic pressure and somewhat in their ability to regulate sodium, and hence to survive in the more saline waters. From the haemolymph osmotic data it seems that death occurs when the osmotic response curve crosses the isosmotic line. In C. bi$da this occurs at A, 0*9O”C, and in C. expletu at A, l+ZZ”C. These values correspond to water from Long Lake with concentrations of 20,500 and 28,000 p-mhos/cm, at 25°C respectively. These values are consistent with the field data which show that C. bz;fda occurs in waters with conductivities in the range of 27 to 20,000 p-mhos/cm (at 25°C) and C. expleta in waters up to 30,000 CL-mhoslcm (SCUDDER,1969a). Since the osmotic responses of haemolymph and urine, and the data on concentrations of sodium, potassium, and chloride in the haemolymph indicate that there is no significant difference between C. bi$da and C. expleta at the low salinities, one might conclude that both species are able to live equally well in fresh water. However, the field data show that while C. bi$da is present and breeds in fresh water C. expZeta does not (SCUDDER,1969a, b). The absence of the more saline tolerant C. expleta from freshwater is not correlated with an inability to regulate the internal milieu in this environment over a short period of time, and the species can certainly live in fresh water for a considerable period (SCUDDER, 1969b). C. expleta is like many other saline tolerant forms that are capable of hyperosmotic regulation in fresh water or solutions of low concentration (NEMENZ, 1960; POTTS and PARRY,1964; PHILLIPS, 1970). For example, an ability to survive and regulate in fresh water has been demonstrated for the saline tolerant Aedes cumpestris D. & K. (PHILLIPS and MEREDITH, 1970), though this species has only been taken in rather saline habitats and does not occur in numerous neighbouring fresh water localities (SCUDDER,1969b). Again, while Procladius nubifer lives in
894
G. G. E. SCUDDER, M. S. JARIAL,ANDS. K. CHOY
saline lakes, it can survive in fresh water, and yet is recorded to have disappeared from Lenore Lake in Washington when the T.D.S. dropped from 6-O to 2.3 g/l. (LAUER, 1969). It would seem that the absence of C. expleta from various fresh-water
lakes is not likely to be due to an inability to survive in these waters. Ecological factors such as predation, availability of suitable food, interspecific competition, and other ecological factors may be important (BEADLE, 1943). As noted by LAUER (1969), in these species that live in inland saline waters “the ecological interactions may be most decisive in determining distribution in water of lower salinity, with physiological tolerance limits of the species becoming the more important determining factor at higher salinity”. Acknowledgements-This research was supported by a grant to G. G. E. S. from the National Research Council of Canada. We wish to thank Dr. A. B. ACTONand Dr. J. E. PHILLIPSfor helpful suggestions and for reading the manuscript, and Miss J. MEREDITH for technical assistance. REFERENCES
BEADLEL. C. (1939) Regulation of the haemolymph in the saline water mosquito larva Aedes detritus Edw. g. exp. Biol. 16, 346-362.
BEADLE L. C. (1943) Osmotic regulation and the faunas of inland waters. BioZ. Rew. 18, 172-183.
BEADLE L. C. (1969) Osmotic regulation and the adaptation of freshwater animals to inland saline waters.
ve’erh.int. Verein. Limn. 17, 421-429. Untersuchungen zur C)kologie der Wasserwanzen, mit besonderer Beriicksichtigung der Brackwasserwanze Sigura Zugubris Fieb. Zool. Jb. (Physiol.) 58, 365-432. CROGHAN P. C. (1958) The osmotic and ionic regulation of Artemia salina (L.). 3. exp. Biol. 35, 219-233. LAUJXR G. J. (1969) Osmotic regulation of Tanypus nubifer, Chironomus plumosus, and Enallagma clausum in various concentrations of saline lake water. Physiol. Zoiil. 42, 381-387. NEMJWZ H. (1960) On the osmotic regulation of the larvae of Ephydra cinereu. J. Insect Physiol. 4, 38-44. PHILLIPS J. E. (1970) Apparent transport of water by insect excretory systems. Am. 2001. 10, 413-436.
CLAUSA. (1937) Vergleichend-physiologische
PHILLIPSJ. E. and MEREDITH J. (1969) Active sodium and chloride transport by anal papillae of a salt water mosquito larva (Aedes campestris). Nature, Lond. 222, 168-169. Porrs W. T. W. and PARRYG. (1964) Osmoticand Ionic Regulation in Animals. Pergamon, Oxford.
RAMSAYJ. A. and BROWNR. H. J. (1955) Simplified apparatus and procedure for freezingpoint determinations upon small volumes of fluid. r. scient. Instrum. 32, 372-37.5. RAMSAYJ. A., BROWNR. H. J., and CROGHAN P. C. (1955) Electrometric titration of chloride in small volumes. g. exp. Biol. 32, 822-829. SCUDDER G. G. E. (1969a) The distribution of two species of Cenocorixa in inland saline lakes of British Columbia. J. ent. Sot. B.C. 66, 32-41.
SCUDDER G. G. E. (1969b) The fauna of saline lakes on the Fraser Plateauin British Columbia. Verb. int. Verein. Limnol. 17, 430-439. SHAWJ. (19.55) Ionic regulation and water balance in the aquatic larva of Sialis Zutaria. J. exp. Biol. 32, 353-382.
OSMOTIC ANDIONICBALANCEIN TWOSPECIESOF CENOCORIXA
895
SHAW J. and STOBBARTR. H. (1963) Osmotic and ionic regulation in insects. Adv. Insect Physiol. 1,315-399. STEPHEN W. P. and JOHNSON0. W. (1962) Qualitative changes in insect blood proteins after freezing. r. Kansas ent. Sot. 35, 189-196. SUTCLIPPED. W. (1960) Osmotic regulation in the larvae of some euryhaline Diptera.
Nature, Land. 187, 331-332. SUTCLIPPED. W. (196fa) Studies on salt and water balance in caddis larvae (Trichoptera)-I. Osmotic and ionic regulation of body fluids in ~~~~~~ afinis Curtis. J. exp. Biol.
38, 501-519. SUTCLIFFED. W. (1961b) Studies on salt and water balance in caddis larvae (Triehoptera)II. Osmotic and ionic reguIation of body fluids in Linmphilus stigma Curtis and Anabolia nervosa Leach. J. exp. Biol. 38, 521-530. WIGGLB~WORTH V. B. (1938) The regulation of osmotic pressure and chloride concentration in the haemolymph of mosquito larvae. J. ex$. Biol. 15, 235-247.