Cation concentrations in the haemolymph of the fly Chironomus thummi, during development

CATION CONCENTRATIONS IN THE HAEMOLYMPH THE FLY CHIRONOMUS THUMMI, DURING DEVELOPMENT

OF

KISSU SCHIN and RICHARD D. MOORE Department

of Biological

Sciences, State University of New York. College Plattsburgh. New York, 12901. U.S.A. (Rrceired

12 Jmuary

of Arts and Science.

1977)

Abstract-The concentrations of the haemolymph monovalent and divalent cations have been determined during the development of Chiranarnus thtmi. a fly. The insect maintains a low and rather constant level of sodium and potassium ion throughout most of the fourth instar period until the time of the larval--pupal ecdysis (LL = 87.6mM Na; l0.8mM K: EPP = 77.4mM Na; 11.7 mM K; LPP = 83mM Na: 14.6mM K). During the final period of development. as the pupa apolysis to a pharate adult there is a significant increase in sodium and potassium ion tevels (EA = 149.4 mM Na: 49.6 mM K). This sharp change of the haemolymph environment is coincident with the occurrence of many of the dramatic metamorphic changes in the animal. e.g.. the breakdown of the salivary gland. and the initiation of vitellogenesis, among others. Artificial media containing the same concentrations of ions as the haemolymph enabled the in vitro maintenance of salivary glands for periods of up to 48 to 72 hr. The importance of the present information in studies of chromosoma) puffing and in other cellular activities such as those leading to cell breakdown has been discussed.

INTRODUCTION THE MOST dramatic

events of postembryonic development in insects are those leading to the metamorphosis. In Cllironomus thummi, the salivary gland ceils are transient, functioning in the production of a secretion only until the time of metamorphosis. They finally break down, and ultimately dissolution completes sometime after the termination of the iarvalpupal apolysis (SCHIN and CLEVER, 1968; LAUFER and SCHIN. 1971: SCHIN and LAUFER, 1973). In the salivary gland cells, the processes leading from the early action of hormones to their final breakdown are accompanied by characteristic puffing changes in their giant chromosomes (CLEVER, 1965). It is presumed that at least part of these puffing changes are concerned with cellular activities which include also the breakdown of the cell itself (CLEVER, 1965, 1966). Although various parameters that affect these tissue-specific events have been studied during insect development (SCHIN and CLEVER, 1965, 1968; HENRIK~ON and CLEVER, 1971: SCHIN and LAUFER, 1973). the mechanisms by which these processes are regulated remain largely unknown. Least known of all is the possible role of the haemolymph constituents, particularly the r8le of the ions of the haemolymph that are in direct communication with the salivary gland cell. The haemolymph provides the necessary conditions for the growth and function of the salivary gland and other tissues. It is important, therefore. to study whether the ionic milieu outside of the cell undergoes alterations at the

critical stages during the course of development. The role of electrolytes in the osmotic regulation of the body fluid has long been recognized. The electrolytes have been suggested as playing an important role in development (MORRILL, 1965; MORRILL er ~11..1967: SLACR rt crl.. 1973; DICK and HO-YEN. 1974). in hormone action (MOORE, 1965; MOORE, 1973). and in gene activation (KRBGER, 1963, 1966: MOORE, 1966: MWRE

and

MORRILL,

1976).

In Chironornus the importance of the monovalent (and divalent) ions in the salivary gland function has been recognized, in that the two major ionic species, sodium and potassium. can affect hormone-dependent gene activity. as evidenced by the chromosomat puffing changes (KRUEGER,1963, 1966; KRUGER and LEZZI. 1966; LEZZI and GILBERT, 1970; KRUEGERet (I/., 1973: LEZZI, 1974). Except for short summaries (FIRLING, 1970; FIRLING and KOBILKA, 1976) and reference to unpublished results (KR&ER rr (II., 1973). however. no information is available on any significant changes in the ionic concentration of the haemolymph. which might affect the salivary gland function at some critical stages during the course of development. The present communication deals with the developmental studies of haemolymph ions in Chironomus thunrmi during the stages when the salivary gland cells undergo extensive changes in preparation for the cell breakdown. The main focus of the present study is to provide some baseline data on the haemolymph ions, and thus to aid in the design of suitable culture media which would be beneficial in further investigation: in particular. in uitro investigation of chromoso-

KISSIJ SCHIN AND RICHARDD.

724

ma1 puffing and other cellular functions and activities such as those leading to cell breakdown. MATERIALS

AND

METHODS

Animals Chironomus t/mm& larvae were reared in plastic tanks at 20°C on a food mixture described previously (LAUFER and WILSON, 1970). The criteria used for the classification by developmental stage were external and internal morphology (LAUFER and NAKASE, 1965; TRAVIS and SCHIN. 1976a). In the present study we adopted the terms pharate pupae and pharate adults (HINTON, 1968) in lieu of prepupae and pupae, respectively, used in earlier papers. Determination of the concentrations of sodium, potusGum, calcium, and mugnesium in the haemolymph Animals were washed in deionized water and briefly blotted. The haemolymph was collected into, and is volume measured in, the pre-calibrated polyethylene tubing (Intramedic, PE 10, Becton, Dickinson and Co.) from an opening cut into one of the prolegs while the larvae were immersed in a mixture of non-polar solvents of SOY,, light mineral oil and 20”, polymer oil (Kel-F oil, 3 M Co.). Both ends of the tube containing the haemolymph were then sealed with oil and kept at - 1o’C. These samples were centrifuged under oil in heat-sealed plastic tubes (Intramedic, PE 205. Becton. Dickinson and Co.) at 2,000g. sedimenting cellular contaminants. if any. The supernatant haemolymph samples were then diluted lOOO-fold with deionized water and the protein precipitated with Y,, TCA. After 30min the precipitate was removed and the supernatant was tested for sodium at 589 m/* and potassium at 766 rnp in the specially constructed. ultra-sensitive flame emission photometer, using a hydrogen-oxygen flame. For the determination of calcium and magnesium concentrations. the haemolymph was pooled to approximately 10 ~1 and centrifuged at 2,000g. The supernatants were then diluted IOO-fold with deionized water and assayed for calcium at 423 rnp and for magnesium at 283 rnp in the atomic absorption flame emission spectrophotometer (Jarrell Ash, Division of Fisher Sci. Co.), using an acetylene-air flame.

MOORE

Cannon’s medium (CANNON, 1964) (b), from Grace’s medium (GRACE, 1962) (c), or from Firling’s medium (personal communication) (d). A possible effect of the developmental stage in the culture was tested by incubating the salivary glands of two different age groups (e and d in Fig. 3) in the salt media complemented with other components from Firling’s media. In addition, two basic salt media were used to test a possible developmental importance of the haemolymph cation concentrations in the salivary gland culture, one being medium (a) and the other one having the cation concentrations found in the haemolymph of young pharate adult stage. Criteria used to measure the viability of the glands were the external morphology, and the size of Balbiani Rings (BRs) and nucleoli. Following incubation, at least one out of ten glands was chosen at random for autoradiographic examination and then compared with control glands.

RESULTS The results in Fig. I show that during development of Chironomus systematic changes occur in the sodium and potassium concentrations of the haemolymph during the period from the late third instar to the early pharate adult stage. By far the highest concentration of sodium and potassium is in the late third instar and early fourth instar. Although there may be an increase in the concentration of sodium

Salivary gland culture To test the effectiveness of various media in the culture the larval salivary glands were dissected from the animal in a bacteria-free chamber under aseptic conditions. The glands, with or without attached fat bodies, were incubated in one of the following media: the basic salt media (composed of 87 mM of NaCl. 10.8 mM of KH,PO,. 3.6mM of MgSOL, and 2.2mM of CaCl,. adjusted to pH 6.8) representing the haemolymph salt concentration of fourth instar larval stage (a), the basic salt media complemented with all the other growth-promoting factors. from

EL ML LL EP LP EA 3L Fig. 1. The concentration of sodium (0) and of potassium ) in the haemolymph of Chirorlomus rhummi. The bars, f @, indicate mean + S.E. 3L = late third instar; EL = early fourth instar; ML = mid-fourth instar: LL = late fourth instar; EP = early pharate pupa; LP = late pharate pupa: EA = early pharate adult.

Ion concentrations

in Chiro~rnrrs haemolymph

725

(+41”,,, P > ( +x1,,. P > 0.25) and of potassium 0.101 from the late third instar to the beginning of fourth instar. these changes are not statistically significant. These high early levels are followed by a precipitous change, with the sodium concentration dropping to 1lOmM (P < 0.005) and the potassium concentration dropping to 16.3 mM (P < 0.005) by midfourth instar. The further decline between mid-fourth instar and late fourth instar of the sodium to 87.6 mM and of potassium to 10.8 mM is not significant (P < 0.1). However. compared to the mid-fourth instar. the concentration of sodium and of potassium of both the early pharate pupa and the late pharate pupa are significantly decreased (P < 0.05). The data suggest that the concentration of both monovalent cations have remained essentially constant, with the sodium concentration near 83 mM and the potassium near 12 mM from late fourth instar to late pharate pupa. However, with the completion of the larvalpupal ecdysis. immediately following the late pharate pupal stage. where overt cell breakdown occurs. the period of stable concentration of haemolymph sodium and potassium ends. By the next stage. early pharate adult the concentration of sodium has risen sharply to 149 + 13 (SE.) mM (P < 0.005) and that of potassium has risen to 49.6 + 15.6 (S.E.) mM

EIIE

I

I

EL

1

ML

I

I

LL

EP

-

LP

Fig. 2. The M&a ratio (above) and the concentration of magnesium (0) and of calcium (0) in the haemolymph of Chironomus thummi.

(P < 0.05).

A brief inspection of Fig. 1 might suggest that the ratio of sodium to potassium ions remains constant. This is indeed the case during the two periods. late third instar to early fourth instar. and late fourth instar to late pharate pupa, when the concentration of each cation remains essentially constant. However. the ratio of sodium to potassium changes significantly from a value of about 3.7: 1 during the first of these two periods to a value of about 6.X during the second period (P < 0.005). Concentrations of calcium and of magnesium in the haemolymph were determined between early fourth instar and late pharate pupa. During the period from early fourth instar to mid-fourth instar. when both sodium and potassium are changing most rapidly. the level of calcium in the haemolymph remains unchanged (P > 0.05). but the concentration of magnesium increases by about 40”,, (P < 0.05). Between mid-fourth instar and late fourth instar, the drop in magnesium ( - 16”,,. P > 0.05) and the rise in calcium P > 0.10) in the haemolymph is not statisti( + F’,,. callv significant. This is the beginning of the period during which the haemolymph levels of sodium and of potassium are held constant. From early pharate pupa to late pharate pupa. the haemolymph level of magnesium remains essentially unchanged. however ;I srgnificant drop ( -St”,,, P < 0.05) brings the concentration of calcium back to the level observed in the early fourth instar. Then between early pharate pupa and late pharate pupa. the stage just before haemolymph sodium and potassium again increase. calcium drops to 2.92 f 0.16 (SE.) mM (P < 0.02) (Fip. 2). The ratio of the magnesium concentration

to the calcium concentration in the haemolymph do not show a consistent pattern. fluctuating around a value of approximately 2: 1 (Fig. 2. above).

The basic salt medium which we have designed on the basis of the haemolymph data of the late IVth instar larva have been tested on the salivary gland (Fig. 3) and ovaries. In addition, the basic salt medium was used with and without organic nutrients. adopted from Cannon’s medium (CANNON. 1964). Grace’s medium (GRACE. 1962), and Firling’s medium (personal communication) for salivary gland culture. The results show that there is a steady decrease with respect to time in the percent of surviving glands in all media tested. For glands from fourth instar larvae, it is notable that during the first six hours of culture. the percentage survival in the basic salt media(a) is almost as good as in the three media containing additional components (b. c, d in Fig. 3). By 24 hr. however, the survival in the basic salt medium is significantly less (P < 0.05) than that in media b and d. This decrease in survival in the basic salt medium. compared to the other three media, is even more manifest by 48 and 72 hr (P < 0.005 in all three). During the entire period, there has been no evidence of difference in the survival rate in media b. c, and d containing the basic salt medium plus added components. At each period of incubation (3. 6, 24. 48. and 72 hr), the survival rate of glands at

KISSU SCHIN ANDRICHARDD. MOORE

3

b

‘-I-

24

48

71

HOURS AFKR INCUBATION

Fig. 3. The survivability of the salivary glands in the culture media. The salivary glands were incubated in one of the following media: the basic salt medium (composed of 87mM of NaCl, 10.8mM of KH2P04. 3.6 mM of MgS04, and 2.2 mM of CaCl,. adjusted to pH 6.8) representing the haemolymph cation concentration of fourth instar larva (a), the basic salt medium complemented with all the other growth-promoting factors: from Cannon’s medium (b), from Grace’s medium (c), or from Firling’s medium (d). Also, groups of the salivary glands from very late pharate pupae were incubated in the salt media complemented with other growth-promoting components from Firling’s medium (e). Sucrose was used to adjust

the osmolarity

as necessary.

very late pharate pupal stage has been significantly less than those in the fourth instar larval stage (d, P < 0.005). Though the results are not always consistent, it appears that a larger number of glands from late pharate pupae have undergone glandular degeneration within 6 hr. while more larval glands have remained functionally intact for an extended period, some surviving up to three days. It is also notable that, to a large extent. the rate of success of the salivary gland culture for periods longer than one day depends at least on three common limiting factors in addition to the age of the tissue: aseptic culture condition, care in dissection of the completely intact salivary gland, and removal of the secretion material from the culture media. In most cases injury to the salivary gland cell during dissection has been one of the major factors for the low success rate of the salivary gland culture in these media. Normally, a portion of thin fat body or fat body-like structures are attached to one or two terminal parts of the salivary gland. Attempts to isolate the salivary gland during dissection commonly induce strain to these structures which result in injury to one or more cells adjacent to these structures. Often the injury that occurs to one cell rapidly spreads to the adjacent cells, resulting in an eventual breakdown of the whole salivary gland, a phenomenon consistent with communication of cells with each other (LOEWENSTEIN et al., 1965). Throughout these experiments, we have, in addition to qualitative autoradiography. used methylene blue or the degree of pigmentation change of the cell as a marker to distinguish between the intact cells and injured cells. We have already reported that membrane changes (indicated by color change of the cell or diffusion of dye into the cell) induced by the injury to one cell resulted rather frequently in the collapse of Balbiani Rings (BRs) in adjacent cells, with the

The bars indicate

the S.D.

BRs of the ones nearest to the injured cell most rapidly affected (SCHIN et al., 1975). It is however not known whether the collapse of BRs represents inhibition of RNA synthesis or rapid dissociation of RNP particles from BR regions. Attempts to study the developmental significance of the cation concentrations in the salivary gland culture have been made also by incubating the tissue in the salt media representing the cation concentrations at two developmental stages. It is of interest to note that after 6 hr of incubation in the salt medium reflecting the haemolymph cation concentrations of the young pharate adult only 27.3 (SE. k 4.7)“,, of fourth instar larval glands remained intact. However, during the same 6 hr period, but in the medium reflecting the haemolymph cation concentrations of fourth instar larvae twice as many, 53.6 (SE. + 5.1)“; of fourth instar larval glands remained intact. In contrast to the larval glands, only 9.2 (S.E. f 1.1)“” and 8.2 (S.E. i l.l)“& of glands from very late pharate pupae survived for longer than 6 hr in the salt medium reflecting the haemolymph cation concentrations of two different developmental stages, late fourth instar and young pharate adult stage, respectively. Although it is difficult to determine to what extent the cation concentration may influence the salivary gland function, the data suggest at least that the age of the tissue and the salt concentration reflecting the haemolymph of the animal are the two important, possibly intimately-linked factors in the salivary gland culture. These experiments show that in vitro studies of the salivary gland, using culture media, must be designed with very careful consideration given to the limiting factors mentioned. We have found that the most successful method of keeping the salivary gland intact for a long period is to maintain the gland with the fat bodies that are

Ion concentrations

in Chironomu.s haemolymph

attached to the tissue. We have not been able to establish the relationship between these structures and the salivary glands. Studies of chromosomal puffing pattern change in the medium are obviously necessary in order to test for the effectiveness of the culture media. Such studies are under way. Developing pharate adult ovaries have also been incubated in the media for one to three days. During this period the ovaries have continued undulation movement. However, we have not been able to find any evidence that vitellogenesis continues in such media. Ovarian culture in the in vitro culture media seems somewhat more difficult than the salivary gland culture, since ovarian development depends on the uptake of blood protein. We have already shown that yolk granule maturation is associated with the uptake of haemoglobin (TRAVIS and SCHIN, 1976a. b).

DISCUSSION Knowledge of changes in haemolymph constituents. such as the inorganic ions, is of paramount importance to the developmental studies of the Ay, Chirono~lls. since the haemolymph is the direct extracellular environment. from which the cell can acquire substances and receive signals needed to function, Changes in haemolymph ion concentration might thus represent a source of information to alter the physiological function of many tissues and organs surrounded by this environment. In Chirononu the salivary gland is a tissue for which the importance of inorganic ions is recognized. in that the ions may affect hormone-dependent chromosomal puffing (KR~ER, 1963. 1966). The significance of this is that variations of the Na/K ratio, perhaps well within the physiological range, might affect gene actions that depend on juvenile hormone and/or ecdysone titers (KR&ER and LEZZI, 1966; LEZZI and GILBERT, 1970; KRUEGERet ul., 1973). On the basis of in uitro work, it has been speculated that ecdysone may act by decreasing the intracellular Na/K ratio, while juvenile hormone may increase the relative or absolute sodium concentration (KRBGER, 1963, 1966). Currently it is assumed by some workers that these insect hormones might support the differential gene actions by affecting transport of these ions across the nuclear membrane (KRUGER et al., 1973). This assumption could be strengthened by carefully controlled studies using in vitro media simulating the haemolymph. Reconstitution of the haemolymph environment is certainly most desirable. Our data clearly show that at earlier developmental stages in the third and early part of the fourth instar, sodium and potassium ion concentrations are significantly higher and vary considerably more among larvae within animals near to the third-fourth instar larval ecdysis. After this period there is a precipitous drop in the sodium and potassium ion concentrations. Following the mid-fourth instar larval stage, the in-

777

sect consistently maintains a low level of sodium and potassium ion from late fourth instar through late pharate pupal stage until the time of the larval-pupal ecdysis. This is the period during which many of the visible chromosomal puffs are induced in in riw and in vitro under the influence of ecdysone. If we assume that the puffing changes that occur during this period are associated with the changes in the ratio of intracellular Na/K. the driving force of such change must be other than changes of haemolymph sodium and potassium level. The change in the sodium and potassium content in the post-larval period which occurs during the period from late pharate pupa to early pharate adult. is perhaps most notable, inasmuch as this change occurs almost in concommittance to. or by coincidence with. the overt cell breakdown of the salivary gland, intestinal muscles. and with the commencetnent of vitellogenesis among others (SCI-IIN and CLEVER. 1965. 196X: KLOETZEL and LAL:FI:R. 1969. 1970; LAUFER and SCHIN, 1971: HENRKSON and CLEVER. 1972: SCHIN and LAUFER. 1973; TRAVIS and SCHIN.1976a. b). We have not been able to correlate this change to the many drastic metamorphic e\.ents occurring during this period: nor have we been able to determine whether breakdown of tissues might directly affect haemolymph ion concentration. In view of the fact. however. that considerably higher numbers of the larval salivary glands undergo rapid degeneration in the simulated haemolymph of pharate adult stage. it is possible to assume that such a change of ionic level could affect the breakdown of the salivary glands in in riro as well. Our data do not significantly deviate from previously reported spot-checked data (KR~~~;ERet trl.. 1973). showing that haemolymph sodium content is consistently higher than potassium. Our results do show. however. a difference from those data. in that the potassium level in the haemolymph remains much higher throughout the course of development. yielding a much lower positive Na,‘K ratio. an average of nearly one fourth of the value reported previously (KR&ER et (I/.. 1973). It is possible that differences between the present results and those referred to by KR&ER et rrl. (1973) may be due to differences in the media and other culture conditions. The importance of having a proper Na:K ratio in insect cell culture from Blccedh and Leclcopli
728

KISSU SCHIN

AND

mechanism for these ions must exist. The fact that the concentration of both sodium and potassium remained relatively constant during three developmental stages is a clear indication of the existence of such a regulatory mechanism for these ions. Indeed, the rather small scatter (variance) for not only sodium and potassium, but also for calcium and for magnesium at any one stage indicates the regulatory mechanism has little variance between animals. Perhaps most striking is the systematic change in the concentration of sodium and of potassium which occurs as a function of the stage of development. It is, of course, logically possible that the reverse correlation also holds namely that the stages of development may somehow be a function of the changes in concentration of these two ions. Although the pattern of change of the divalent cations is not as clear cut as that of the monovalent cations, the regulation of these ions seems equally certain. It is perhaps of some interest that a change occurred in divalent cation concentration before each of the two major changes in monovalent cation concentration. Haemolymph magnesium increased before the drop in both sodium and potassium and magnesium and calcium both decreased before the final rise in haemolymph sodium and potassium. The present data do show that experiments with in vitro media must be carefully conducted, reflecting the ionic concentration of the same stage. Though the survival of the tissue in the media is limited, it is noteworthy that the tissue can be maintained for long periods in the medium whose ionic composition is comparable to haemolymph of the appropriate developmental stage. This suggests that the changes that occur in the concentration levels of the haemolymph ions are significant, possibly a necessary factor in the morphogenesis of Clziro~ornus. Acknowledgemenrs~The authors are indebted to Drs. J. NOLAN and D. LEE at SUNY Plattsburgh. for their kind criticism and advice in the preparation of the manuscript. We thank particularlv Dr. H. LAUFER at the Universitv of Connecticut, Storrs. who helped develop the technique of haemolymph collection, and who made many constructive suggestions. We acknowledge Dr. V. MUNK for allowing us to use specially designed chamber for Chironomus culture. Thanks are also due to Dr. C. FIRLING at the University of Minnesota, Duluth, for providing us with unpublished results, and to C. EDMISTON and S. SEEHOLZER for their capable laboratory assistance. This work was supported in part by grants from the SlJNY Research Foundation. from the NIH (AM 17531) and from the N.E. Section of the New York Heart Association.

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