Mechanisms of excretion and ion transport in invertebrates

Prévia do material em texto

17. Mechanisms of excretion and ion 
transport in invertebrates 
Department of Biology, McMaster University, ’* 0 O I Hamilton, Ontario, Canada 
C H A P T E R C O N T E N T S 
Ion Transport and Osmoregulation in Invertebrates: Functional 
Morphology and Cellular Mechanisms 
sponges, and cnidarians 
Proton pumps in protozoans 
Water expulsion vesicles and contractile vacuoles in protozoans, 
Nephridia and coelomoducts: embryology and terminology 
Nephridia 
Metanephridia in annelids 
Interactions of molluscan transporting epithelia: gill, mantle, 
and excretory system 
Coelomoduct-derived renal organs 
Antenna1 and maxillary glands in crustaceans 
Onychophoran segmental glands: similarities to arthropod 
Coxal organs and salivary glands in arachnids and centipedes 
Malpighian tubules of insects 
Malpighian tubules of myriapods 
The insect hindgut 
Labial glands 
Ion transport by the integument and gut in annelids 
Calciferous gland of earthworms 
Eversible water uptake organs 
Crustacean gills 
Tube foot epithelium of echinoderms 
Water transport independent of production of osmotic gradients 
by epithelial ion transport: vapor uptake by the desert cock- 
roach 
excretory organs 
Gut-derived renal organs 
Other ion-transporting structures 
Excretion in Invertebrates 
Nitrogenous wastes 
NH, 
Urea 
Uric acid 
Other purines and pyrimidines 
Free amino acids 
Alkaloids 
Organic anions 
Organic cations 
Magnesium and sulfate 
Crustaceans 
Insects 
Molluscs 
Structure, composition, and functions of metal-containing 
Insects 
Molluscs 
Storage and deposit excretion 
granules 
Crustaceans 
Mobilization of calcium from storage granules 
Catabolism of insect neurohormones by Malpighian tubules 
Roles of arthropod excretory systems and molluscan mantle in 
acid-base regulation 
Regulation of hemolymph pH in insects by Malpighian tubules 
Transport of bicarbonate by Malpighian tubules 
Contributions of crustacean antenna1 glands to acid-base 
Contributions of molluscan mantle to acid-base regulation 
Active transport of sugars 
Transport of cardiac glycosides 
Passive permeability to metabolites and toxins 
and intestinal epithelia 
regulation 
Future Research 
THE DIVERSITY OF EXCRETORY AND OSMOREGULATORY 
MECHANISMS in the more than thirty-five invertebrate 
phyla is extraordinary, and encyclopedic coverage of 
this area is not feasible in a single chapter. In general, 
this chapter focuses on animals and systems that have 
been most amenable to study at the cellular level. It 
deals primarily, therefore, with studies that make use 
of in vitro preparations, isolated tissues, cells, or mem- 
brane vesicles, where modern techniques of electro- 
physiology, pharmacology, biochemistry, and molecu- 
lar biology could be applied. 
Many reviews of ion transport and excretion in 
individual groups of invertebrates have been published. 
Excretion, ionoregulation, and water balance in insects 
have been examined by Noble-Nesbitt (338), Phillips 
et al. (377), Bradley (34,35), and Cochran (79). Com- 
parable reviews are available for crustaceans (294), 
molluscs (28, 298, 382), and annelids ( 3 5 4 ~ ) . In addi- 
tion, several excellent monographs provide a broader 
perspective. Water balance in terrestrial arthropods 
has been discussed by Edney (100a), excretory and 
osmoregulatory changes required for life in the terres- 
trial environment by Little (254, 255), evolution of 
homeostatic mechanisms for osmotic and ionic regula- 
tion in marine invertebrates by Prusch (397), storage 
excretion in invertebrates by Brown (44), and aspects 
1208 HANDBOOK OF PHYSIOLOGY-COMPARATIVE PHYSIOLOGY 
of ion transport relating to absorption of atmospheric 
water vapor by Machin (266) and O’Donnell and 
Machin (347). Comparative physiology texts by With- 
ers (546) and Prosser (388) provide excellent introduc- 
tions to excretion and ionoregulation in vertebrates 
and invertebrates. 
This chapter is divided into two main sections. The 
first concerns the structures and cellular mechanisms 
involved in ion transport and osmoregulation in inver- 
tebrates. The second concerns the roles of these struc- 
tures in the excretion of wastes and toxins, in acid-base 
regulation, and in the reabsorption of useful molecules 
from excretory fluids. 
ION TRANSPORT AND OSMOREGULATION IN 
INVERTEBRATES: FUNCTIONAL MORPHOLOGY AND 
CELLULAR MECHANISMS 
Water Expulsion Vesicles and Contractile Vacuoles in 
Protozoans, Sponges, and Cnidarians 
Freshwater protozoans are hypertonic to their medium 
and must offset the tendency for osmotic water entry 
and resultant swelling. In protists with cell walls, a 
hydrostatic pressure is established to counterbalance 
the osmotic pressure exerted by intracellular osmolytes. 
The alternative mechanism involves an efflux of water 
to offset the osmotic influx of water across the plas- 
malemma. The subcellular organelles which produce 
the efflux are called contractile vacuoles or water ex- 
pulsion vesicles (WEVs). WEVs slowly fill with fluid 
and periodically expel their contents from the cell. The 
majority of WEVs are surrounded by a system of fine 
(20-50 nm) membranous tubules or vesicles, known 
as the spongiome. Many ciliates have distensible re- 
gions of the spongiome, called ampullae, which dilate 
as they accumulate fluid from the WEV or other parts 
of the spongiome. 
The WEV complex in ciliates, such as Paramecium, 
consists of an extensive and complex system of 
membrane-delimited spaces. The radial shape of the 
complex is maintained by a series of ribbons formed 
from microtubules, which act as cytoskeletal ribs, radi- 
ating from a permanent pore and holding the mem- 
branes in place (160). As the contractile vacuole ex- 
pands, its membrane is forced against the inner margin 
of the pore cylinder, thereby facilitating membrane 
fusion and the opening of the pore (5) . Both excretion 
and collapse are probably facilitated by the hydrostatic 
pressure within the cell forcing the vacuole contents 
out through the open pore. The vacuole, ampullae, and 
collecting canal membranes assume tubular form when 
internal volume is at a minimum. Studies based on 
serial sections suggest that the contractile vacuole mem- 
brane collapses following systole into an extensive 
three-dimensional array of anastomosing membrane 
tubules, not distinct vesicles (5). The collapsed vacuole 
membrane forms flattened cisternae, which fenestrate 
and transform into small tubules with time. Importan- 
tly, there is no ultrastructural or biochemical evidence 
for a matrix of microfilaments lining the vacuole mem- 
brane. The term “contractile” may be misleading, 
therefore, and the forces which transform the flattened 
sheet of membrane into a complex mass of anastomos- 
ing tubules may be an intrinsic property of the mem- 
brane itself. In the absence of a microfilamentous en- 
casement of the vacuole, Allen and Fok (5) suggest 
that the mechanism of water expulsion involves fluid 
volume increase and fluid flow within periodically in- 
terconnected tubular membrane systems, which cycle 
between tubular and planar forms as internal volume 
is periodically increased and reduced. 
Considerations of the energetic costs of cell wall 
biosynthesis and volume regulation indicate that in 
growing cells the energy input for WEV operation is 
much less than for wall synthesis, a t least in terms of 
the minimum thermodynamic requirements of the two 
processes (419). When plausible mechanisms are con- 
sidered, the two energy costs are broadly comparable 
for cells with relatively high hydraulic conductivity 
values (10-’4m.s-’-Pa-1). For lower and more rep- 
resentative water permeabilities (Lp= m-s- ’ . 
Pa-*), the energy cost of WEV function is only about10% of the mechanistic energy cost for cell wall pro- 
duction. Reduction or cessation of growth favors cell 
wall production over WEV operation. In starved cells, 
running the WEV system demands most of the energy 
from the resting metabolic rate, thus providing a strong 
selection pressure for volume regulation by means of 
cell wall production during dormancy or encystment. 
However, WEVs are a necessary concomitant of flagel- 
lar activity and the consequent absence of a cell wall. 
In fact, the energy requirements of WEV operation, a 
necessary correlate of flagellar locomotion, use more 
of the cells’ energy than the flagella themselves. In 
addition, increased intracellular osmolarity causes a 
directly proportional increase in energy input required 
for cell wall biosynthesis but a more than proportional 
increase, perhaps as much as a squaring, in the cost of 
WEV operation. There is thus a strong energetic ratio- 
nale for the reduction in intracellular osmolality in 
freshwater vs. marine protozoans. 
WEVs are also important components of the adap- 
tive strategies of euryhaline protozoans, such as the 
green flagellate Chlamydomonas pulsatilla (1 67). WEV 
activity decreases as salinity is increased from 0% to 
1570, but the WEV persists as a collection of small 
CHAPTER 17: EXCRETION AND ION TRANSPORT 1209 
vesicles or tubules in cells grown in more concentrated 
media. The time required to empty one cell volume 
increases from about 20 min in 1% seawater to 600 
min in 15% seawater. Growth rates are comparable 
over this 30-fold range of WEV activity, indicating that 
the cost of WEV operation at these salinities is low. 
There are three readily identifiable events associated 
with WEV functioning: diastole (filling); systole, the 
reduction in size of the WEV; and expulsion, the release 
of vacuolar fluid from the contractile vacuole to the 
outside (362). The most accepted mechanism of filling 
involves osmosis. Accumulation of an osmolyte with 
the WEV complex creates an osmotic gradient and 
resultant water flow into the complex. The current 
working hypothesis is that ion transport across the 
membranes of the spongiome creates the necessary 
osmotic gradient. The WEV complex remains fully 
functional when cells are placed in deionized water, so 
resorption and subsequent recycling of ions must be 
postulated. Production of hypoosmotic fluid by a WEV 
was first demonstrated by Schmidt-Nielsen and 
Schrauger (448a). Osmolality of subnanoliter fluid 
samples collected from Amoeba proteus by micropunc- 
ture was determined by freezing point depression. For 
amoebae acclimated to a medium with a mean osmol- 
ality of 7 mOsm.kg-', protoplasm osmolality was 101 
mOsm-kg-' and vacuolar fluid osmolality was 32 
mOsm kg-'. Schmidt-Nielsen and Schrauger (448a) 
suggested that fluid secreted into the WEV is initially 
isoosmotic with the protoplasm and that hypoosmotic 
fluid is produced by a subsequent process of solute re- 
absorption. 
Filling of the WEV and solute resorption and expul- 
sion may occur at different sites within the complex or 
at the same site whose function changes with time. For 
example, water permeability of the membranes must 
be low at the time or place of solute resorption to 
prevent a backflux of water into the cytoplasm. Simi- 
larly, the WEV membranes probably have a low perme- 
ability to water, to permit storage of hypoosmotic fluid. 
Amoebae require effective ionoregulatory systems 
not only to reduce the effects of passive transmembrane 
diffusion of ions and other solutes but because cellular 
ion composition is also perturbed by pinocytosis. Dur- 
ing this process, small quantities of the surrounding 
medium containing proteins, amino acids, and other 
solutes are taken up by means of membrane infolding. 
Studies on the pathogenic species Entamoeba histolyt- 
ica indicate that each cell takes up about 30% of its 
own volume each hour by pinocytosis. Since the para- 
site is exposed to NaCl concentrations exceeding 100 
mM in growth media or the serum of the host, the 
NaCl load resulting from pinocytosis far exceeds that 
resulting from diffusive influxes across the plasma 
membrane (12b). Cells lose NaCl when pinocytosis is 
inhibited, suggesting that excess Na+ resulting from 
pinocytosis is transferred from the vesicles into the 
cytoplasm and then out of the cell. Sodium content 
within pinocytotic vesicles has been measured in amoe- 
bae loaded with Percoll, which is taken up in pinocy- 
totic vesicles and gives a strong silicon signal when 
freeze-dried sections are analyzed by X-ray microanaly- 
sis. Na+ content of Percoll-containing vesicles declines 
with the age of the veiscles; the half-time for this 
decrease is about 45 min. Overall, it is estimated that 
about 70% of the Na+ taken up by pinocytosis is 
removed by transport across vesicle and plasma mem- 
branes and that about 30% is regurgitated by exo- 
cytosis (12a). 
Pinocytosis can be stimulated by addition of any of 
a variety of cationic inducer substances to the external 
medium. The inducer causes formation of channels 
from the surface of the cell into the cytoplasm (400). 
Calcium plays an important role in the control of this 
process. In Ca2+-free medium, pinocytotic uptake of 
radiolabeled sucrose does not occur. The inducer sub- 
stance Alcian blue (0.01%) increases pinocytotic up- 
take of sucrose as external calcium concentration is 
increased up to a maximum of 0.1 mM. 
Calcium appears to be involved in two distinct 
phases of pinocytosis (396, 400). The initial stage 
probably involves displacement of Ca2+ from nega- 
tively charged sites on the cell surface by the inducer. 
About 18% of the total of 4.6 mM Ca2+.kg-l cells is 
associated with the cell surface (401). Binding of the 
inducer substance increases membrane permeability to 
solutes, including Ca2 + . Cytoplasmic Ca2 + activity is 
normally maintained at a low value by an active pro- 
cess (395), and an influx of Ca2+ in response to inducer 
binding may produce a transient and localized increase 
in Ca2+ beneath the cell surface. Contraction of fila- 
ments associated with the cell surface may be stimu- 
lated by this increase and may bring about surface 
invagination, channel formation, and subsequent for- 
mation of pinocytotic vesicles (395). Evidence for Ca2+ 
involvement in this second stage of pinocytosis has 
been provided by experiments utilizing the Ca2+ iono- ' 
phore A23187. In the absence of inducer substances, 
addition of A23187 increases Ca2+ influx and the 
uptake of radiolabeled sucrose (395). Membrane per- 
meability to sucrose is very low in the absence of pino- 
cytosis. 
There may be different pinocytosis-inducing sites on 
the cell surface. Both Alcian blue and Na+ induce 
pinocytosis, but they associate in different ways with 
the cell surface of A. proteus. Sodium binds loosely, 
and the amount associated with the surface of the 
amoeba is a linear function of the external Na+ con- 
1210 HANDBOOK OF PHYSIOLOGY-COMPARATIVE PHYSIOLOGY 
centration (398). In contrast, the binding of Alcian 
blue can be described by a simple adsorption isotherm, 
with saturation at an external dye concentration of 
about 0.3 mM. 
It is worth noting that Ca2+ also plays a role in 
the related process of phagocytosis, which consists 
of uptake of particulate matter. Calcium influx and 
phagocytosis in A. proteus can be induced by chemo- 
tactic peptides, and both processes can be blocked by 
CaZ+ channel blockers, such as verapamil and flunara- 
zine. Calcium channel blockers have no effect on Ca2+ 
influx under control conditions, suggesting that chemo- 
tactic peptides activate specific Ca2+ channels in the 
cell membrane (403). 
In general, cells swell when WEV output is reduced 
by anoxia or application of metabolicinhibitors. WEV 
output decreases transiently if the osmotic pressure of 
the medium is raised and increases transiently when 
the external medium becomes more dilute. In addition 
to these short-term changes in WEV output, the long- 
term adjustment of protists to osmotic shock involves 
adjustments in the concentrations of intracellular os- 
molytes. Because of the perturbing effects of large-scale 
changes in the predominant intracellular inorganic 
ions, such as N a + and K + , on enzyme structure and 
function (558), long-term changes in intracellular con- 
centrations of what are known as “compatible solutes” 
(558) are used for adjustment of intracellular osmol- 
ality. The green flagellate C. prrlsatilla withstands high 
salinities by accumulating glycerol (2), whereas the 
ciliate Miarniensis avidus adjusts intracellular osmol- 
ality through changes in the concentrations of free 
amino acids, especially alanine, glycine, and proline 
(207a). These three amino acids account for 73% of the 
total free amino acid concentration of 317 m M + kg- in 
cells maintained in 100% seawater. Free amino acid 
content is decreased by 24% in 25% seawater and 
increased by 22% in 200% seawater. Changes in free 
amino acid content are completed within 20 min of a 
salinity change. Amino acids are presumably mobilized 
from proteins or other bound states in cells adjusting 
to increases in salinity and may be polymerized into 
proteins or bound in some manner during adjustments 
to lower salinity (207a). 
The WEV functions not just in osmoregulation but 
also in regulating levels of specific ions. Maintenance 
of hyperosmolality in M . avidus, for example, may be 
necessary to provide water for the contractile vacuole, 
which may function in the elimination of metabolic 
wastes and possibly Na+ (207a). A role of the WEV 
in N a + regulation has also been suggested in other 
protozoans. For example, there is a transient increase 
in cytoplasmic sodium content when WEV activity of 
the ciliate Tetrahymena pyriformis is arrested (234). 
Similarly, sodium concentration in WEV fluid of the 
amoeba Pelomyxa carolinensisin is higher than in the 
cytoplasm (426). In A. proteus, the fluid remaining in 
the WEV after solute reabsorption may be Na+-rich. 
Measurements of ion concentrations and membrane 
potentials indicate that K + is actively accumulated 
from the medium and that N a + is actively extruded 
(399). Active K + influx was suggested by the decline 
in cellular [K+] in response to metabolic inhibition and 
the observation that cellular K+ concentration exceeds 
that in the Nernst equilibrium with the membrane 
potential. Net efflux of N a + against an electrochemical 
potential gradient occurs when Ca2+ is added to cells 
in an Na+-rich medium. I t appears that N a + perme- 
ability is low in the presence of Ca2+ and that a 
sodium pump establishes the N a + gradient under such 
conditions. In low CaZ+ media, the membrane perme- 
ability to sodium is sufficiently high that the outwardly 
directed sodium pump does not maintain a measurable 
gradient (399). It has been suggested that sodium 
pumping may be associated with WEV activity (394a). 
The effects of ionophores, inhibitors of sodium trans- 
port, and changes in the bathing medium sodium con- 
centration on WEV function are difficult to interpret. 
The Na+,K+-ATPase inhibitor ouabain decreases the 
frequency of WEV activity in ciliates (104), but several 
studies indicate that Na+,K+-ATPase does not occur 
in protozoans (7). Elevation of cytoplasmic sodium by 
addition of ouabain, the sodium ionophore monensin, 
or Na+-loaded liposomes or by elevation of external 
sodium concentration (104, 119) induces a decrease in 
WEV activity, a t odds with the suggestions that water 
extrusion is coupled to that of sodium (382). 
One explanation offered for these apparently contra- 
dictory results is that changes in intracellular Na + also 
affect intracellular Ca2+ activity (119). WEV activity 
decreases when external Ca2+ concentration increases 
or decreases relative to the level in the Pringsheim 
solution used to culture the cells (103). Addition of the 
calcium ionophore A23187 to a solution containing 
0.85 mM Ca2+ increases WEV activity in a dose- 
dependent manner. 
Bergquist (1 6) suggests, from experiments with in- 
hibitors, that the control of WEV contraction fre- 
quency is mediated by the calcium-binding protein 
calmodulin. Calmodulin is located a t the site of the 
permanent pore in Tetrahymena (494), suggesting that 
WEV contraction frequency is mediated by a peripher- 
ally located mechanism. 
Contractile vacuoles are common among protists, 
particularly freshwater forms, and are found in fresh- 
water sponges (42), where they also act as intracellular 
pumps responsible for cell volume maintenance. Not 
until the early 1990s they were described in metazoans. 
CHAPTER 17: EXCRETION AND ION TRANSPORT 1211 
active transport. Uptake of both proline and glucose 
across the plasma membrane of Leishmania donouani 
are coupled to proton influx (564,565). Both symport- 
ers are electrogenic and are driven, therefore, by differ- 
ences in both pH and electrical potential established 
by the electrogenic proton pump. 
Malaria parasites of the genus Plasmodium extrude 
H + across the plasma membrane by means of a P-type 
H+-ATPase. Plasmodium spend much of their life cycle 
within the erythrocytes of the vertebrate host and must 
overcome the problems imposed by a low Ca2+, low 
Na+ environment (497). The plasma membrane proton 
pump establishes a membrane potential of about -90 
mV and a pH difference of less than 1 unit. The 
proton motive force drives inward movement of Ca2+, 
possibly through an electrogenic antiporter involving 
three or more H+ ions per Ca2+ ion (231). Glucose is 
transported from the cytoplasm of the erythrocyte into 
the parasite via a glucose/H+ symport (498). 
Transmission electron micrographs reveal a unique 
“zipper-like organelle” in the apical endoderm of the 
stolons of hydroids, including Hydructinia symbiolon- 
gicarpus (446). Evidence from transmission electron 
microscopy and video microscopy suggests that zipper- 
like organelles are, in fact, contractile vacuoles, but 
their functions are very different from the contractile 
vacuoles of protists and sponges. Zipper-like organelles 
absorb and release gastrovascular fluid from a common 
lumen, thereby contributing to exchanges of gases, 
inorganic ions, and organic solutes. Zipper-like organ- 
elles also control gastrovascular flow through effects 
on endoderm volume and, hence, lumen diameter. Vac- 
uolization of the zipper-like organelles closes the lumen 
of the gastrovascular canal, thereby blocking gastrovas- 
cular flow. In contrast, when the endoderm is little 
vacuolized, its thickness is reduced and the stolon 
lumen diameter correspondingly increased (446). 
Proton Pumps in Protozoans. Pinocytotic vesicles in E. 
histolyticu acidify to pH 5.5 immediately after forma- 
tion and maintain this pH as they age. Low concentra- 
tions of bafilomycin A, inhibit this acidification, sug- 
gesting that a vacuolar-type (V-type) proton ATPase 
pumps H+ into the vesicles (12a). I t has been suggested 
that a V-type ATPase also pumps H + out of the cell 
and that Na+ extrusion is driven indirectly by the 
plasma membrane proton pump (12a). Presumably the 
V-type H+-ATPase supplies the driving force for extru- 
sion of Na+ by an Na+/H+ antiporter. 
Studies of ion transport systems may provide clues 
as to the evolution of ionic pumps. It has been sug- 
gested that both V-type and phosphorylated 
intermediate-type (P-type) H + -ATPases were present 
in the plasma membrane of proteukaryotes and that 
the former pump provided the driving force for second- 
ary H+-coupled transportof other ions and solutes 
(291). V-type ATPases may have become restricted to 
internal organelles, such as lysosomes and Golgi bod- 
ies, after incorporation of mitochondria, and P-type 
ATPases may then have come to play a predominant 
role on the plasma membrane. Later in evolution, 
V-type ATPases may have “returned” to the plasma 
membrane in a variety of vertebrate and invertebrate 
cells, such as insect epithelia (12a). In contrast, the 
presence of a V-type H+-ATPase in the plasma mem- 
brane of Entamoeba may represent an archaic feature 
since this species is considered to be a very simple 
eukaryote which probably branched off early in evolu- 
tion. It lacks mitochondria, peroxisomes, rough endo- 
plasmic reticulum, and Golgi apparatus, for example. 
In other parasitic protozoans, a P-type proton H+- 
ATPase situated in the plasma membrane appears to 
provide the driving force for H + -coupled secondary 
Nephridia and Coelomoducts: Embryology and 
Terminology 
Embryological criteria form the basis for two major 
categories of excretory system. Nephridia are tubules 
of ectodermal origin, which develop in a centripetal 
pattern; they are first formed as invaginations of exter- 
nal surfaces and then grow inward. In contrast, coe- 
lomoducts are tubules of mesodermal origin, which 
develop centrifugally; they are first formed as out- 
growths of internal tissues and then grow outward. 
Nephridia are found in platyhelminths, nemerteans, 
rotifers, some gastrotrichs, and polychaete annelids. 
Coelomoducts are characteristic of onychophorans and 
arthropods. On the basis of embryology, Goodrich 
(136) proposed that the excretory organs of adult 
molluscs are coelomoducts, not nephridia, and that 
they were created initially for gamete transport and 
then subsequently modified for excretion. However, 
the close relationships between molluscs and annelids 
in terms of developmental patterns and methods of 
coelom formation, the occurrence of protonephridia in 
the trochophore larvae of molluscs, and the discovery 
of metanephridia in the monoplacophoran Neopilina 
guluthea probably validate use of the term “nephridia” 
for molluscan excretory organs. Certainly, most mod- 
ern texts of invertebrate zoology use it. 
Two other systems for osmoregulation are found in 
nematodes and winged insects. In insects, water and 
salt balance are maintained by the combined effects of 
secretion by the Malpighian tubules, which open into 
the intestine at the junction of the mid- and hindguts, 
and reabsorption by the hindgut. Malpighian tubules 
are also found in myriapods, arachnids, and tardi- 
1212 HANDBOOK OF PHYSIOLOGY-COMPARATIVE PHYSIOLOGY 
grades but have probably evolved independently in the 
latter two groups. In nematodes, a ventral gland cell 
in the pseudocoelom is unfortunately referred to as the 
“excretory cell.” In fact, this cell provides enzymes 
required for shedding the cuticle at ecdysis (89). In 
the codworm nematode Pseudoterranova decipiens, the 
primary site of osmoregulation appears to be the hypo- 
dermis of the body wall (120). Osmoregulatory abilities 
of P. decipiens may be adaptive because larval forms 
are exposed during the life cycle to the blood of cope- 
pods and isopods, intestinal contents, blood and mus- 
cles of cod, and stomach and intestinal contents of 
seals. Intact worms or cylindrical sacs of body wall 
formed by removal of intestine and pseudocoelomic 
fluid do not gain weight for up to 24 h in hypotonic 
media. In the presence of metabolic poisons, both 
whole worms and sacs gain weight in hypotonic media. 
Structural studies suggest that the sites of osmoregula- 
tion may be small cells embedded in the syncytial 
hypodermis, referred to as “hypodermal glands” or 
“bacillary band cells” (557). Two observations suggest 
a minor role for the intestine in osmoregulation: the 
volume of fluid passed from the anus increases when 
worms are maintained in hypotonic media and the 
mass of worms in such media increases slightly if the 
tail is ligatured (120). In addition, osmotic properties 
of the pseudocoelomic fluid may dampen or mitigate 
the effects of water flux into the coelom. Pseudocoelo- 
mic fluid consists of two functional compartments. 
Experiments with 3H20 indicate that water is more 
readily available for exchange in one compartment 
than in the other. I t is suggested that the slowly ex- 
changing compartment may consist, in part, of proteins 
which bind water, perhaps reducing its osmotic activ- 
ity (121). 
Nephridia 
Nephridia are organs which produce urine by first 
filtering extracellular fluid and then modifying the fil- 
trate. Filtration involves the selective separation of 
solutes from water as the extracellular fluid passes 
through the filter. Gradients in hydrostatic or osmotic 
pressure may be used to drive filtration, and the term 
“ultra filtration” denotes the retention of colloidal sol- 
utes. In addition, secretion has been shown to play a 
role in urine production in the nephridia of molluscs 
(384), arthropods (429), and leeches (563). 
There are two basis types of nephridium in aquatic 
invertebrates. A metanephridium consists of an excre- 
tory duct which opens to the outside of the animal 
through a nephridiopore and into a coelomic cavity 
through a ciliated funnel called the nephrostome. In 
contrast, a protonephridium does not open internally 
but is blind-ended. One or more tubular cells, called 
“terminal cells,” contain both the cilia, which extend 
into the nephridial compartment, and a wickerwork, or 
weir, between the extracellular space and the nephridial 
compartment [tubule lumen). Terminal cells are called 
solenocytes if nonciliated, flame cells if multiciliated 
with basal nuclei, or flame bulbs if multiciliated with 
lateral nuclei (545). O n the assumption that nephridia 
are homologous, Goodrich (136) proposed that proto- 
nephridia are primitive to metanephridia and that both 
are excretory organs derived from the ectoderm. Rup- 
pert and Smith (439) have presented a functional model 
of nephridia which downplays the importance of germ 
layer origins. Both types of nephridium share the fol- 
lowing features: (1) two fluid-filled extracellular com- 
partments are present, one enclosing the other (Fig. 
17.1), and (2) a filter separates the compartments. 
Pressure is highest in the outer compartment in the 
case of protonephridia (Fig. 17.1B) and in the inner 
compartment in the case of metanephridia (Fig. 17.1A). 
The application of these terms can be appreciated 
by comparison with the mammalian nephron, in which 
Bowman’s capsule forms the outer coelomic compart- 
ment and the glomerular capillaries form an inner 
vascular compartment. Positive pressure within the vas- 
cular compartment drives fluid across the glomerular 
basal lamina, which functions as a selectively perme- 
able filter. The filtrate flows from Bowman’s capsule 
into a duct, the kidney tubule, where the filtrate is mod- 
ified. 
Metanephridia are also interposed between a coelom 
and the exterior of the body (Fig. 17.1C). The coelom 
encloses blood vessels, and constriction of the vessel 
walls increases internal pressure, driving fluid through 
the layer of podocytes, which overlies the vascular 
basal lamina. By analogy with vertebrates, a region of 
the blood vascular system is functionally similar to the 
glomerulus, the coelom is comparable to Bowman’s 
capsule, and the duct of the metanephridium is analo- 
gous to the tubule of the vertebrate nephron (439). 
A metanephridium can be defined, therefore, as any 
nephridial system involving muscle-mediated filtration 
of vascular fluid into a coelomic cavity and reabsorp- 
tion by an open duct (439). 
In contrast, a protonephridial system involves cilia- 
mediated filtration of extracellular fluid intothe ne- 
phridial lumen and reabsorption by the nephridial duct 
(Fig. 17.10). Selective reabsorption of proteins by en- 
docytosis into protonephridial duct cells indicates that 
protonephridia play a role in excretion as well as 
osmoregulation (477). 
By extension, one would expect metanephridial sys- 
tems to be limited to invertebrates with a blood vascu- 
lar system and protonephridia to be more common in 
CHAPTER 17: EXCRETION AND ION TRANSPORT 1213 
i e r 
modi f icat ion 
- rnetanephridlum 
FIG. 17.1. Functional model of protonephridial and metanephrid- 
ial systems. Direction of fluid flow is indicated by arrows. Two 
designs for filtration nephridia are shown (A, B ) . Fluid flows from 
inner to outer compartment when PI exceeds Pz. Fluid composition 
is adjusted subsequently by the modifier. C: Metanephridium. Trans- 
verse section of a generalized coelomate. Contraction of peritoneal 
musculature elevates blood vascular pressure, thereby filtering fluid 
animals without a blood vascular system. An important 
corollary is that protonephridia are found in small or 
flat animals (larval stages, flatworms) or in larger ani- 
mals (polychaetes, priapulids) if there are no septa to 
impede convective mixing of coelomic or pseudocoelo- 
mic fluids. In polychaetes, all but one of the 14 families 
with protonephridia lack a well-developed blood vas- 
cular system (439). 
Both proto- and metanephridia rely on initial forma- 
tion of urine by ultrafiltration and subsequent tubular 
reabsorption of solutes and water. Epithelia lining the 
ducts of both types of nephridium have apical cilia and 
microvilli, apical coated pits and vesicles, endosomes, 
and lysosomes. 
An important linkage between the two types of ne- 
phridium is evident in studies of metamorphosing in- 
vertebrate larvae. Podocytes of metanephridia and the 
terminal cells of protonephridia may well be alternate 
expressions of the same basic cell type (439). In the 
larvae of the polychaete Sabellaria cementarium, larval 
solenocytes are not lost but transformed into podocytes 
on the wall of the lateral esophageal blood vessels. The 
terminal 
w e i r 
- c i l i u m 
across peritoneal podocytes. Coelomic fluid is subsequently modified 
by metanephridial duct. D: Protonephridium. Schematic transverse 
section through a generalized metazoan lacking blood vessels. Activ- 
ity of terminal cilium (or cilia) creates a pressure difference, which 
drives fluid across terminal weir. Filtration occurs as fluid crosses 
extracellular matrix of weir, and fluid is subsequently modified by 
protonephridial duct. Redrawn from Ruppert and Smith (439). 
occurrence of protonephridia in larvae of many animals 
with metanephridia as adults does not necessarily imply 
that protonephridia are primitive. A protonephridium 
may simply be an alternative design, perhaps more 
efficient for small animals without a circulatory sys- 
tem (439). 
Metanephridia in Annelids. Although a few species of 
polychaete are found in freshwater or terrestrial envi- 
ronments, the vast majority are marine osmoconform- 
ers. Oligochaetes and leeches, which have probably 
derived from oligochaete stock, are more capable of 
significant hyperregulation. Both chloride uptake 
across the skin (94a) and the production of a hypotonic 
urine contribute to osmoregulation. 
In the “open” nephridia of polychaetes, such as the 
feather duster worm Sabella pavonina, the nephro- 
stome cilia move coelomic fluid into the nephridial 
canal (223). Ultrafiltration probably occurs in the peri- 
esophageal capillary network, so the coelornic fluid can 
be thought of as a primary urine. Modification of urine 
composition then occurs as the urine passes down the 
1214 HANDBOOK OF PHYSIOLOGY-COMPARATIVE PHYSIOLOGY 
length of the nephridium. Some polychaetes produce 
only isoosmotic urine (for example, Glycera dibranchi- 
ata), but in others, such as Nereis diversicolor, the 
urine becomes hypotonic in lower medium salinities, 
suggesting ion reabsorption from the primary urine. 
In S. pavonina, coelomic fluid osmotic pressure is 
generally 100 mOsm hypertonic to the external me- 
dium over the range 900-1,500 mOsm. Sodium con- 
centrations in coelomic fluid are roughly equal to those 
of seawater, and K + concentrations are up to 144% 
of seawater values; but amino acid concentrations, 
mostly glycine, are about 35 mM. Final urine is always 
about 5 % hypertonic to coelomic fluid and 12% hyper- 
tonic to seawater, and most of these changes occur 
across the terminal section. Micropuncture data indi- 
cate that the final urine contains higher concentrations 
of sodium and markedly higher concentrations of po- 
tassium relative to the coelomic fluid. Comparisons of 
urinary outflow rate and perfusion inflow rate of iso- 
lated perfused nephridia of S. pavonina indicate that 
there are no significant movements of water across the 
nephridial wall. 
Nephridia in polychaetes also reabsorb amino acids 
from the primary filtrate (223), helping to maintain 
the high free amino acid content of the internal fluid 
characteristic of many marine invertebrates. Free 
amino acids contribute to coelomic fluid hypertonicity, 
relative to the surrounding seawater, and the amino 
acids which are compatible osmolytes (558), such as 
glycine and proline, predominate. The carrier-mediated 
reabsorption of a-amino isobutyric acid by the ne- 
phridial epithelium of S . pavonina is characterized by 
a K,, of 3.7 mM and a V,,, of 0.234 nmol.min-’.ne- 
phridium-’. An apical site for the carrier has been 
suggested (222). Approximately 80% of total uptake 
is sodium-dependent (224), and the concentration of 
N a + in the apical medium which can sustain full 
transport is quite low (17 mM) relative to the N a + 
concentration in the nephridial lumen (420 mM). This 
implies that the normal intracellular concentration of 
N a + may be lower still and that the N a + affinity 
of the uptake system is very high. Sodium-dependent 
transport processes in amino acid uptake by the gut in 
invertebrates are discussed in detail by Wright and 
Ahearn (see Chapter 16, this Handbook). 
Nephridia of S . pavonina also accumulate cholesterol 
and cholesterol esters in the form of intracellular lipid 
droplets. These are found in the infranuclear space of 
the nephridial epithelial cells (225). The sterols may be 
accumulated as stores for the other cells or for oocyte 
production. The large quantities found in the proximal 
cells may indicate that lipids are recovered from the 
primary urine derived from the coelomic fluid. In the 
distal cells, cholesterol may be an excretory product 
since the cytoplasm is discarded from the apocrine cells 
and eliminated in the urine. 
In oligochaetes such as the earthworm, the nephro- 
stome of the nephridium also opens into the coelomic 
space, so the primary urine is presumably isoosmotic. 
NaCl accounts for about 50% of the osmotic pressure 
of the coelomic fluid, and organic substances, such 
as amino acids, probably account for much of the 
remainder. Micropuncture experiments indicate that 
salt reabsorption in the distal half of the nephridium 
reduces the total osmolality of the urine by about 
80% (410). 
In the class Hirudinea (leeches), the excretory system 
is arranged quite differently from that of the two other 
annelid classes, the polychaetes and oligochaetes. In 
this group, the connection between the nephrostome 
(“ciliated funnel”) and the rest of the nephridial tubule 
has been lost. The term “metanephridium” may be 
inappropriate, therefore, since urine formation in this 
group involves secretion by nephridial lobe cells in a 
system of canaliculi which runs into the nephridial 
canal. 
Mechanisms of salt and water regulation in leeches 
have been examined mostextensively in the European 
medicinal leech Hirudo medicinalis (reviewed in ref. 
561). In fresh water, leeches produce urine which is 
hypotonic to the blood (31 vs. 189 mOsm.kg-’) but 
which nonetheless leads to salt loss. Leeches compen- 
sate for this ionic loss to the surrounding water by 
active salt uptake across the integument (see later under 
Other Ion-Transporting Structures). Invasion of brack- 
ish waters, which are more concentrated than the 
blood, or ingestion of blood meals both represent forms 
of hypertonic stress. Under these conditions, salt excre- 
tion rates are augmented. 
Isolated nephridial preparations of H. medicinalis 
permit analysis of ion transport in vitro. The current 
model of ion transport is based on analysis of fluids 
collected by micropuncture and electrophysiological 
measurements during ion substitution and/or drug ap- 
plication (Fig. 17.2). Both K + and CI- are actively 
transported from blood to primary urine. A 
furosemide-sensitive N a + :K+ :2C1- cotransport step, 
stimulated during diuresis following a blood meal, 
appears to be present on the basolateral cell membrane, 
along with a ouabain-sensitive N a + K+ exchange pump 
and a Ba2+-sensitive K + conductance. CI- exits the 
cell down a favorable electrochemical gradient, either 
through channels or in a KCl cotransport step (562). 
Transepithelial N a + transport may occur paracellu- 
larly, in response to a favorable transepithelial poten- 
tial (TEP) of about -7 mV, lumen-negative. Ouabain 
blocks urine flow, but amiloride M ) has no 
effect, suggesting that neither amiloride-sensitive N a + 
CHAPTER 17: EXCRETION AND ION TRANSPORT 1215 
-7mV 
-63 mV 
Lumen 
67 rnM Na+ 
51 m M K * 
I 19 mM C1- 
Apical --n-+t+ 
O r n V 
Blood ? 
Na+ 
I26 mM Na' 
s mM K+ 
36 mM C1- 
E lec t rochemica l p o t e n t i a l s ( t ransep i the l i a l ) 
(Blood+ Lumen) 
ENa = -23 mV ( favo r ing ) 
EK = +52 mV (opposing) 
ECI = -37 mV (opposing) 
FIG. 17.2. Summary of ionic compositions of blood and secreted fluid, suggested mechanisms of ionic 
transport, and electrochemical potentials for secretion of Na+, K+, and CI- by the leech canaliculus. 
In this and all subsequent figures, ATP-dependent pumps are indicated by circles labeled +ATP; 
secondary active transport systems (co- or countertransport) by open circles, and passive conductive 
pathways (for example, channels) by arrows through gaps in plasma membrane. 
channels nor Na+/H+ exchange is integral to the mech- 
anism of ion-coupled fluid secretion. This situation is 
in contrast, therefore, to the effects of amiloride on 
fluid secretion by blood-feeding insects such as Rhod- 
nius (283). There is also evidence for modification of 
the primary urine; ion recycling permits production of 
a dilute final urine, just as insect blood-feeders such as 
Rhodnius (described below, see Gut-Derived Renal 
Organs) resorb KCl to adjust the osmolality of the 
urine. 
Quantitative differences are apparent in the salt- 
transporting abilities of Hirudo relative to the North 
American leech, Macrobdella decora. Hirudo tolerates 
salinities up to 400 mOsm * kg-' and is found in brack- 
ish waters, whereas M . decora is less tolerant. After 
hypertonic crop loading, salt excretion increases 60- 
fold in Hirudo and sevenfold in Macrobdella and urine 
flow increases eightfold within 30 min in Hirudo and 
only threefold over longer periods in Macrobdella. In 
sum, Hirudo absorbs fluid more rapidly from the crop, 
the primary urine-forming cells of the nephridia have 
a higher transport capacity, and salt is reabsorbed more 
rapidly from the central canal of the nephridium (522). 
Mechanisms controlling urine volume are indepen- 
dent of those controlling urine osmolality in both H. 
medicinalis and M . decora. The volume of urine ex- 
creted depends on blood volume and is not correlated 
with blood osmolality. Continuous monitoring of ex- 
tracellular C1- concentration has been implicated in 
the control of nephridia in Hirudo. This form of con- 
trol is in contrast to the involvement of stretch- 
receptors in blood-feeding insects. The chloride concen- 
tration of leech blood is much less than the cation 
concentration (36 vs. 130 mM), and the anionic deficit 
is made up by organic acids (560). Chloride concentra- 
tion of leech hemolymph rises 2.5-fold after a meal of 
1216 HANDBOOK OF PHYSIOLOGY-COMPARATIVE PHYSIOLOGY 
blood, which is hypertonic and contains C1- as the 
predominant anion. Chloride also rises after emersion 
or movement from fresh to brackish water. 
Changes in hemolymph chloride concentration may 
be detected by a nephridial nerve cell associated with 
each nephridium. The cell body lies on the urinary 
bladder, the dendrites extend into the nephridium, 
and the axon runs into the corresponding segmental 
ganglion via the nephridial nerve. The burst rate of 
the nephridial nerve cell changes fourfold, without 
adaptation, in the physiological range between 40 mM 
and 90 mM C1-. Nephridial nerve cells do not respond 
to changes in blood pressure, osmolality, pH, hemo- 
lymph organic acid concentrations, blood volume, or 
stretch of the bladder wall. Intracellular recordings 
suggest that a high CI- conductance in combination 
with active transport of CI- could form the basis of 
the chloride sensitivity of neuronal activity. It is worth 
noting that neurons of other invertebrates exposed to 
hypoosmotic conditions commonly show a swelling- 
induced increase in potassium permeability (5 13). Al- 
though the postsynaptic targets of the nephridial nerve 
cell are unknown, it seems likely that this ion receptor 
cell affects salt excretion through effects on urine con- 
centration (523). FMRF-amide immunoreactivity has 
been identified in the nephridial nerve cell, and applica- 
tion of low ( 1 . 7 X l O - ' O ) but not high ( 1 . 7 ~ 1 0 - ~ ) 
levels of exogenous FMRF-amide increases urine flow 
rates more than twofold (523a). Control of ion and 
fluid secretion in leeches and insects is quite different. 
Relatively little use is made in leeches of blood-borne 
effectors, such as diuretic hormones, in contrast to 
insects. Instead, control of fluid secretion by the leech 
excretory system seems to be exerted through innerva- 
tion of the nephridia. 
Interactions of Molluscan Transporting Epithelia: Gill, Mantle, 
and Excretory System. Both cephalopod and gastropod 
molluscs filter the primary urine through the heart 
wall, either into the pericardial cavity or directly into 
the kidney (383). The nephrostome typically opens into 
the pericardial cavity via a renopericardial duct. In 
cephalopods, the metanephridia or kidneys bear en- 
larged regions called renal sacs. The kidney is con- 
nected to the pericardial coelom by the renopericardial 
canal and empties into the mantle cavity via the 
nephridiopore. The renal canals receive pericardial fil- 
trate via the renopericardial canal but also receive 
secretions from the renal appendages, which are 
formed from evaginations of the wall of the branchial 
vein as it passes through the renal sac. As the branchial 
heart beats, deoxygenated blood collected from the 
body is drawn through the renal appendages and filtra- 
tion occurs across their thin walls into the sac. Selective 
reabsorption of useful solutes occurs within the coe- 
lomic cavity even before filtrate enters the renal sacs 
(298). 
Pericardial fluid is also formed by ultrafiltration from 
the hemolymph in some bivalves. In the Japanese oyster 
Crassostrea gigas, filtration pressure oscillates between 
about 32 mm H 2 0 and -4 mm H 2 0 and both protein 
concentration and average molecular weight are re- 
duced in the filtrate relative to the hemolymph (179). 
The coefficient of filtration, which is affected by factorssuch as thickness, pore diameter, and pore geometry, 
is 4.5 x ml.s- ' .cm-2 mm Hg-', intermediate 
between the values for this parameter in frog and 
human glomeruli. In contrast, Pierce (379) found no 
evidence of ultrafiltration in six other species of lamelli- 
branch, including Anodonta and Modiolus. 
Comparative aspects of kidney function have been 
studied in nereticid gastropod molluscs, which inhabit 
marine, freshwater, and terrestrial environments (252). 
The marine species Nerita fulgtrrans is unable to osm- 
oregulate and does little ionic regulation. The kidney 
does not control the ionic or osmotic composition of 
the hemolymph. In contrast, the glandular part of 
the kidney of the freshwater neritid Neritina latissirna 
resorbs salts. Terrestrial species also produce a dilute 
urine, though the site of ion resorption is unclear. 
Changes in kidney reabsorbtive capability in marine 
vs. freshwater and terrestrial species are not correlated 
with gross structural reorganization. 
Freshwater and euryhaline bivalves also maintain 
blood osmolality and ionic concentrations significantly 
above those of the surrounding water, though they are 
less effective osmo- and ionoregulators than fish or 
crustaceans, Blood-medium differences of over 200 
mM for both N a + and C1- are maintained by blue 
crabs (171), for example, while the corresponding val- 
ues for bivalves are, a t most, 25 mM (388). 
The combined actions of several distinct epithelia 
contribute to iono- and osmoregulation in molluscs. 
Studies of the osmoregulatory role of ATPases in bi- 
valves have indicated adaptive changes in Na +,K+- 
ATPase activity in gill, mantle, and kidney in response 
to changes in external salinity (90, 91, 137, 303, 443). 
The euryhaline marsh clam Rangia cuneata is unusual 
among bivalves in that it is capable of significant hyper- 
osmotic regulation of the body fluids (359). Na+,K+- 
ATPase activity is higher in mantle than in other tis- 
sues, and acclimation to low salinities is associated 
with adaptive increases in Na+,K+-ATPase activity in 
the mantle epithelium but not the gill. The number of 
ouabain-binding sites does not increase during acclima- 
tion to low salinity, suggesting that increased pump 
activity involves activation of preexisting pump sites in 
the mantle epithelium. 
CHAPTER 17: EXCRETION AND ION TRANSPORT 1217 
rich (MR). Clusters of MR cells are also found in 
the water channel epithelia, along with many 
mitochondria-poor cells. The lamellar filaments of the 
surface filaments, with the exception of the lateral 
ciliated cells, also contain mitochondria-poor cells. His- 
tochemical analyses indicate that MR cells of the water 
canals and water channel contain high levels of CA 
and cytochrome oxidase. It seems likely, therefore, that 
the MR cells of the water canal and water channel 
epithelia are the primary salt-transporting cells (215). 
An understudied aspect of ion transport in molluscs 
is the potential role of mucoid concretions in renal 
structures. Andrews (6) has suggested that adsorption 
of ions onto the surface of the concretions, which are 
held in close proximity to the apical cell membrane of 
nephridial gland mucous cells, may result in higher 
local ion concentrations and lower energetic costs for 
adsorption (6 ) . In cephalopods, Schipp et al. (447) 
suggest that ion resorption may be aided by mucoid 
concretions in the renal appendages. 
In general, it appears that both the gills and the 
excretory system contribute to hyperregulation in gas- 
tropods and bivalves. Gills are involved in active ion 
uptake, and ion losses are minimized by reabsorption 
from the urine. Such capabilities are presumably lack- 
ing from the cephalopods, which are exclusively 
marine. 
Active uptake mechanisms (K, 0.2-0.3 mM in two 
species) are physiologically appropriate for uptake 
from pond water containing 0.05 mM K+. Surpris- 
ingly, freshwater bivalves are very sensitive to potas- 
sium levels in the bathing media; concentrations of 
0.6-1 mM impair survival in as little as 24 h. Excess 
uptake of K+ under these conditions leads to an in- 
crease in extracellular K+ levels and a depolarization 
of excitable tissues, with toxic effects. Other freshwater 
invertebrates, fish, and amphibians are less sensitive to 
K+, so short-term treatment with KCl may be an 
effective means for reducing populations of pest species 
of mussel (94b). 
Adaptive changes in epithelial ATPase activities have 
also been identified in mesogastropods (501). Na+,K+- 
ATPase activity does not vary with salinity in the 
marine osmoconformer Littorina littorea, and enzyme 
activities are significantly higher in freshwater species 
Viviparus contectus and Pornacea canaliculata. These 
two species hyperregulate in brackish or fresh waters 
by active uptake of Na+ and C1- across the gill and 
resorption of these ions from the urine (253). Much of 
the ion resorption in Viviparus appears to be accom- 
plished by the nephridial gland, a discrete region of 
the kidney’s dorsal wall (251). 
Carbonic anhydrase (CA) activity in gill and mantle 
tissue also increases when osmoregulating bivalve mol- 
luscs are subjected to osmotic stress (173). However, 
the levels of enzyme activity in bivalve gills are more 
than 1,000-fold lower than in blue crab gills, 300- 
900 mM C02.mg protein-*.min-l. The CA inhibitor 
acetazolamide impairs hyperregulation of Na+ and 
C1- levels when ambient osmolality is reduced. Gener- 
ation of H+ and HCO; by the action of CA on C 0 2 
presumably provides counterions for the uptake of 
Na+ and Cl - , respectively. Thus, exchange processes 
appear to act in conjunction with ion-activated AT- 
Pases (508,509). Possible actions of an HC0;-ATPase 
in C1- regulation by the gill epithelium of freshwater 
bivalves have also been suggested (95). 
Histochemical and ultrastructural studies have impli- 
cated particular cell types in ion uptake by the gills of 
freshwater unionid mussels (215). Each of the four gills 
consists of an ascending and a descending lamella, with 
a central water cavity. Septa oriented parallel to the 
filaments partition this internal chamber into parallel 
tubes, referred to as “water channels.” Water entering 
the mantle cavity passes through water canals, which 
run at right angles to the long axis of the filaments, 
and into the central water channel. The openings of 
the water canals facing the mantle cavity are termed 
“ostia.” The water canals account for about 38% of 
the total surface area of the gills. All of the epithelial 
cells lining the lateral water canals are mitochondria- 
Coelomoduct-Derived Renal Organs 
In crustaceans, the coelomoducts open at the base of 
the second antenna or second maxilla and are termed 
“antennal glands” and “maxillary glands,” respec- 
tively. In arachnids, the coelomoducts open in the sixth 
segment and are termed “coxal glands.” 
Antennal and Maxillary Glands in Crustaceans. In general, 
the antennal organs of malacostracan crustaceans show 
anatomical and physiological similarities to the verte- 
brate nephron (428). Their role appears to be mainte- 
nance of hemolymph volume by filtration and excretion 
of osmotically gained water. Antennal glands are com- 
prised of an end sac, an excretory canal, and a short 
duct leading to the exterior. In decapods, the end sac 
consists of a saccule for collection of fluid and a spongy 
labyrinth for reabsorption. A tubule connects the laby- 
rinth and bladder. 
In amphipods, the antennal organs receive hemo- 
lymph at relatively high pressure, almost directly from 
the systemic heart. The pressure differential between 
hemocoel and the sum of coelomosac hydrostatic and 
colloid osmotic pressure may drive filtration across 
the coelomosac wall. The limiting filtration barrier is 
probablythe basal lamina of the coelomosac (217). 
1218 HANDBOOK OF PHYSIOLOGY-COMPARATIVE PHYSIOLOGY 
In crayfish, barnacles, and amphipods, hydrolysis of 
protein in formed bodies may generate local osmotic 
gradients, driving fluid across the coelomosac wall 
(427, 503, 531). 
Modification of primary urine occurs in many crusta- 
ceans. As discussed later under Magnesium and Sulfate, 
Mg2+ and can be excreted by the antennal 
glands. In most crabs studied, the ratio of the inulin 
concentration in urine vs. blood (U/B ratio) is greater 
than or equal to unity (430), and water reabsorption 
is a more likely explanation than inulin secretion. In 
most species, the antennal glands do not play a major 
role in osmoregulation and ions lost in the urine are 
replaced by uptake across the gills (see later under 
Other Ion-Transporting Structures). The crayfish an- 
tennal gland, however, posseses a long tubule between 
labyrinth and bladder. Salt reabsorption by the tubule 
results in a dilute urine, aiding hyperregulation. Pro- 
duction of dilute urine appears to occur in a morpho- 
logically distinct region of the distal tubule (367), 
where N a + is actively reabsorbed by the Na+,K+- 
ATPase, followed by CI - (206). Active solute reabsorp- 
tion also occurs in the bladder. Antenna1 glands contain 
high activity levels of Na+,K+-ATPase and CA. Hydra- 
tion of COz by the latter ion provides counterions 
(H+, HCO;) for the transport of N a + and CI-. 
The antennal gland appears to be of minimal use in 
salt regulation in several terrestrial crabs (Gecarcoidea 
landii, Cardisoma carnifex, Birgus latro) and is, in fact, 
the major site for loss of salt and water from the 
animal (230). During dehydration, urine flow rate is 
reduced (151). In hydrated crabs, the fluid secreted by 
the antennal gland is slightly hyperosmotic and con- 
tains about the same level of sodium as, and slightly 
more chloride than, the hemolymph. When given access 
to distilled water, however, most of the N a + in the 
urine is not lost. Although the possibility of reingestion 
of urine has been suggested (230), it appears more 
likely (see later under Other Ion-Transporting Struc- 
tures) that crabs such as Ocypode and Birgus reprocess 
urine in the branchial chamber and that ions are reab- 
sorbed across the gill epithelium. 
Onychophoran Segmental Glands: Similarities to Arthropod 
Excretory Organs. Onychophorans are usually consid- 
ered phylogenic relics, possessing both annelid and 
arthropod characteristics. The nephridies typiques oc- 
cur in all but the fourth and fifth segments and are 
characterized by prominent bladders but short seg- 
ments. Modification of the primary urine has been 
deduced from U/P inulin ratios and urine composition 
(64). The filtration rate is 2.5 pl/h and the urine pro- 
duction rate 4.2 pl/h in Peripatus acacioi, indicating 
addition of 1.7 pl water/h by the tubular epithelium. 
The daily filtration and urination rates equal 30% 
and 50% of body water, respectively, comparable to 
aquatic rather than terrestrial animals and consistent 
with the availability of water in the animal’s habitat. 
The coelomic vesicle appears to form the primary urine, 
which is then progressively modified by the tubular 
epithelium and/or the bladders and excretory ducts. 
Long flagella in the nephrostome may force fluid along 
the tubule and produce negative pressures in the coe- 
lomic vesicles, thereby driving ultrafiltration, as in the 
protonephridia. The tubular epithelium reabsorbs so- 
dium, chloride, and calcium but secretes potassium, 
magnesium, and phosphates. At urine p H of 5.3 (64), 
most phosphate is present as acid, suggesting an im- 
portant role for renal structures in the elimination of 
excess protons, as in vertebrates. Measured ions ac- 
count for only half of the osmolality of urine, which is 
isoosmotic (but hypoionic) to the hemolymph. 
The fourth and fifth pairs of nephridia are much 
larger than the nephridies typiques and lack bladders. 
Cells of these two pairs of nephridia produce hypoos- 
motic urine by secondary reabsorption of hyperosmotic 
fluid (65). These cells have poorly developed apical 
microvilli but invaginations associated with mitochon- 
dria on the basal surface. Their structure and function, 
therefore, appear to resemble those of the thick as- 
cending limp of Henle’s loop in the mammalian neph- 
ron and the early distal tubule of the amphibian neph- 
ron. Reduction of lumenal ion concentration by 
reabsorption of hyperosmotic fluid is in some ways 
similar to potassium chloride reabsorption by the lower 
Malpighian tubule of the insect Rhodnius prolixus 
(287). Ion reabsorption is sodium-dependent but rather 
nonspecific with respect to anions; chloride can be 
replaced by nitrate, for example. Inhibition by ouabain 
suggests that a basal Na+,K+-ATPase drives ion reab- 
sorption (65) . 
Coxal Organs and Salivary Glands in Arachnids and Centi- 
pedes 
Coxal organs in argasid ticks. Coxal organs in argasid 
(soft-bodied) ticks are bilateral structures whose ori- 
fices exit between the first and second pairs of coxae, 
which are the limb segments closest to the body. Both 
the volume and ionic composition of the hemolymph 
are regulated by the coxal organs, which work by a 
filtration-reabsorption mechanism. After a blood meal, 
NaCl is actively transported from the gut and water 
movement is coupled osmotically. An increase in hemo- 
lymph hydrostatic pressure initiates ultrafiltration 
across the filtration membrane of the coxal organ (209, 
210). Studies with extracellular space markers indicate 
that access of hemolymph solutes with molecular 
masses < 5 kd is unrestricted. Fluorescently labeled 
bovine serum albumin is trapped within pockets of the 
filtration membrane, suggesting the latter as the site 
of ultrafiltration. The rate and volume of coxal fluid 
production are directly related to the rate and volume 
of fluid absorption by the gut epithelium. High rates 
of absorption across the gut result in high rates of 
NaCl loss due to increased filtration and decreased ion 
reabsorption. However, a low rate of NaCl transport 
and osmotic water flux from gut to hemolymph lowers 
the rate of coxal fluid production, thereby providing 
more time for NaCl to be reabsorbed. Glucose is reab- 
sorbed from the fluid in the lumen of the coxal tubule 
by a phlorizin-sensitive mechanism. Amino acids are 
CHAPTER 17: EXCRETlON AND ION TRANSPORT 1219 
also reabsorbed, and those reabsorbed most effectively 
are in short supply in the blood meal. 
Salivary glands in ixodid ticks. The salivary glands of 
ixodid (hard-bodied) ticks are involved in the elimina- 
tion of excess water and ions during the blood meal 
(Fig. 17.3). Up to 96% of the Na+ and 74% of the 
water in the blood meal is secreted back into the 
host via the salivary glands. Some potassium (16%) is 
eliminated via the salivary glands, but the Malpighian 
tubules eliminate most of the potassium (84%), which 
is then excreted (211-214). The predominant osmo- 
lytes in tick saliva are Na+ and C1-, though there is 
an additional major anion component, possibly HCO J. 
The ion transport mechanisms involved in saliva pro- 
Host I 
FIG. 17.3. Schematic showing routes for ingestion of blood meal and excretion of ions and water by 
an ixodid tick, Derrnacentor andersoni. Redrawn from Kaufman and Phillips (211). 
1220 HANDBOOK OF PHYSIOLOGY-COMPARATIVE PHYSIOLOGY 
duction are not known. Secretion rates for isolated 
glands vary with the chloride concentration of the 
bathing saline, and active chloride transport is sug- 
gested by higher concentrations of CI - in the lumen 
relative to the bathing fluids and an opposing electrical 
gradient of 36 mV. Fluid secretion is also ouabain- 
sensitive, so active exchanges of Na+ and K+ may also 
be involved. Although the glands secrete in K+-free 
media, secretion rates increase up to fivefold in salines 
containing not more than 10 mM K + . Higher K + 
concentrations are inhibitory. 
Water vapor absorption by ticks. The salivary glands 
and/or mouthparts are also involved in the water bal- 
ance of ticks when they are off the host. Although ticks 
are obligate blood-feeders, they spend up to 98% of 
their life cycle off the host and may endure several 
years between blood meals. During these periods, imbi- 
bition of liquid water either does not occur or has 
been shown to be insignificant, and active water vapor 
absorption (WVA) is an important component of the 
tick's water balance. WVA is an energy-dependent 
process which permits net uptake of atmospheric wa- 
ter. The process appears to have evolved independently 
in such diverse groups as tenebrionid beetle larvae, 
chewing lice, psocopterans ("book lice"), thysanurans, 
terrestrial isopods, desert cockroaches, and flea larvae, 
as well as ticks and mites of several different orders. 
Not only does uptake occur at quite different localized 
sites in different arthropods, but mechanisms involving 
epithelial secretion of hygroscopic solutions, elec- 
troosmosis, modulation of the water affinity of hydro- 
philic polymers, and compression of an enclosed vol- 
ume of air to elevate humidity above saturation have 
been proposed (347). 
In ticks, WVA exploits the availability of humid 
microclimates. Net uptake is possible at relative humid- 
ities (RH) above about 90% and at temperatures as low 
as -1"C, suggesting that WVA may be an important 
component of water balance in over-wintering ticks 
(203), as well as during the summer months. Surpris- 
ingly, some species of engorged ixodid and argasid 
ticks absorb water vapor (203,205). The physiological 
advantage of WVA after the blood meal is unclear. 
The structures involved in tick WVA are the salivary 
glands and the mouthparts. A fluid is secreted by the 
salivary glands onto the mouthparts of ticks during 
absorption (436-438). Selective destruction of types I1 
and 111 granular acini of the salivary glands by infection 
with a protozoan parasite (Theileria annulata) does 
not impair uptake, implicating type I agranular acini 
in absorption. Ultrastructural changes of the mitochon- 
dria of certain cell types of the agranular alveoli during 
cycles of dehydration and rehydration support this 
conclusion (326). Moreover, comparison of the struc- 
ture of granular and agranular alveoli of the salivary 
glands at several points during the life cycle indicates 
active vapor uptake when the granular alveoli are 
atrophied or have disintegrated completely, confirming 
the preeminence of the agranular alveoli in WVA (204). 
The role of hypertonic saliva in providing a thermo- 
dynamic gradient driving condensation of atmospheric 
water vapor is currently the subject of controversy. 
Early studies examined the crystalline solids collected 
from the mouthparts of dehydrating ticks. Preliminary 
analysis indicated that the amount of Na+ in the solids 
exceeded that of K+ (437, 438). Analysis of frozen 
sections suggests the presence of hypertonic fluids 
within the salivary ducts, and it has been suggested 
that active water uptake by adult ticks (Amblyomma 
americanum) is a solute-driven process based upon 
production of hyperosmotic fluid, probably by the type 
I agranular acini of the salivary glands (461). Ticks 
engaged in active WVA can be rapidly frozen in hexane 
cooled with liquid nitrogen, then embedded and sec- 
tioned in a cryostat at -20°C. The sections can then be 
placed on a temperature-controlled microscope stage in 
the cryostat and gradually warmed to 0°C in 0.5"- 
2.0"C increments. The temperature at which ice crystal 
movement is observed is taken as an indicator of the 
melting point. As noted below, this method may not 
provide a reliable indication of the melting point. Os- 
molalities calculated from the melting points range 
from 5.4-6.4 Osm- kg-' in the lateral salivarium to 
2.7-4.8 Osmakg-' in the central salivarium, compared 
to 0.54-1.61 Osm-kg- ' in the hemolymph. 
Saliva samples can also be collected after stimulation 
of salivation by light and heat stress (461). However, 
osmometry of nanoliter samples by freezing point de- 
pression indicates that oral fluid osmolality varies from 
0.43 to 5.11 Osm- kg-'. Higher osmolalities corre- 
spond to smaller fluid volumes, raising the possibility 
that osmolality may have been artificially elevated by 
evaporative concentration during exposure of animals 
to the desiccating effects of the heat lamp. 
A very different hypothesis emerges from the studies 
of Amblyomma variegatum by Gaede (122, 123) and 
Knulle (personal communication). The linear relation- 
ship of WVA rate to ambient humidity for ixodid ticks 
suggests that hydrophilic structures and/or substances 
may be involved between threshold (<90% RH) and 
98% RH and that hygroscopic fluids may augment 
vapor uptake but only at very high (>98% RH) hu- 
midities. 
In the study of Gaede (122), measurements of freez- 
ing point depression of saliva and hemolymph collected 
from rehydrating ticks indicate that the osmolality of 
saliva is insufficient to drive vapor uptake at humidities 
less than about 98%. Saliva can be collected by micro- 
CHAPTER 17: EXCRETION AND ION TRANSPORT 1221 
pipette from previously desiccated ticks exposed to 
humidities (93% RH) appropriate for WVA. The fine 
channel which runs between the chelicera and the 
hypostome can be visualized in restrained ticks, and 
the tip of a collecting pipette can be placed between 
hypostome and chelicera and inserted as far as the 
basis capitulum. Fluid samples can be readily collected 
by sucking on a rubber tube connected to the back of 
an oil-filled pipette and exposure of the sample to air 
after collection minimized by filling the tip with oil 
after sample collection. The fine end of the collection 
pipette can then be broken off and the opening sealed 
with melted wax. The colorless saliva is readily distin- 
guishable from hemolymph, which is turquoise. In the 
study of Gaede (122), each sample volume was on 
average 2.2 nl, with a minimum measurable volume of 
0.27 nl. Freezing point depression was determined in 
a custom-built nanoliter osmometer, which was cali- 
brated before and after each sample measurement with 
NaCl standards. The collection pipette and enclosed 
sample were plunged into liquid freon cooled to 
- 11 1°C with liquid nitrogen, then quickly transferred 
to the cooling block of the osmometer, which had 
been preset to a temperature below the suspected 
freezing point of the sample. During a subsequent 
10 min equilibration period, ice crystals were observed 
to coalesce. The sample was gradually warmed 
(0.2"C.min-') and the temperature adjacent the sam- 
ple measured to the nearest 0.01"C with a thermocou- 
ple. The temperature when the last ice crystal disap- 
peared was noted and taken as the melting point. 
Mean osmolality of saliva collected from 41 A . oarie- 
gatum specimens in the study by Gaede (122) was 
470.3 mOsm-kg-' (range 299-770 mOsm-kg-'). 
Mean hemolymph osmolality was 342 mOsm * kg- 
(range 271-472). Both saliva and hemolymph concen- 
trations varied linearly with the degree of predesicca- 
tion in the range of 2.8%-16.5% loss of initial weight, 
and the ratio of saliva to hemolymph osmolality was 
1.39 & 0.24. 
These data suggest that the saliva is not sufficiently 
concentrated to directly drive water vapor uptake since 
uptake at humidities of 93.5% RH corresponds to an 
osmolality of 3,859 mOsm.kg-', five times greater 
than the maximum osmolality of collected saliva. 
Gaede (122, 123) suggests that the agranular salivary 
acini have two functions:(1) storage excretion of sur- 
plus inorganic ions and other solutes during periods of 
desiccation and (2) modulation of the water affinity of 
hydrophilic mouthparts. In other words, the saliva 
may, in fact, promote release of condensed water from 
the mouthparts in a manner analogous to the cyclical 
release of water from the hydrophilic cuticle of the 
hypopharyngeal bladders of the desert cockroach Are- 
nivaga (343; see later under Other Ion-Transporting 
Structures). 
In ticks, the hydrophilic structures may consist of a 
flexible membrane on the dorsal surface of the hy- 
postome. Partial covering of this membrane with teflon 
foil, by placing the foil between hypostome and chelic- 
era, results in lower rates of absorption, and the extent 
of the decrease corresponds to the proportion of the 
membrane surface area covered by the foil (122). It 
appears that the whole hypostome is involved in WVA, 
not just the distal portions. The possible contribution 
of the hydrophilic cuticle is indicated by the rapid 
uptake or loss of water from isolated hypostomes when 
the humidity is varied. 
Coxal organs and the anol system in spiders. Although 
ectoparasites, such as ticks, are adapted to feeding 
on particular mammalian hosts of relatively invariant 
blood composition, fluid-feeding predators, such as 
spiders, must cope with fluid influxes of variable ionic 
composition. For example, the Na/K ration of the 
spider's meal will vary with the proportions of intracel- 
lular and extracellular fluids ingested. The osmolarity 
of spider and scorpion hemolymph is usually between 
about 400 mOsm and 520 mOsrn, well below that of 
seawater but well above that of most insects. Burton 
(5 1) suggests that a high extracellular osmolality may 
match that of the partly digested prey. Na+ and C1- 
are the predominant hemolymph osmolytes in scorpi- 
ons and some spiders, but in other spiders half the 
osmolality is due to organic solutes (51). Replacement 
of Na+ and C1- by organic osmolytes may be an 
advantage in insects and spiders with excretory systems 
which include Malpighian tubules since loss of these 
molecules and the need for reabsorption will be much 
less than in filtration-based systems in crustaceans, 
for example (51, 325). Concentrations of organic and 
inorganic osmolytes are strongly intercorrelated; in- 
creases in "a+] and [K+], for example, are associated 
with increases in the concentrations of organic osmo- 
lytes. Amino acids, for example, can be 50-100 times 
more concentrated in insect hemolymph relative to 
mammalian plasma (539). Since organic acids bind to 
divalent cations, most calcium in the hemolymph is 
bound. Hemolymph calcium activity is of the order of 
1-2 mM, whereas total concentration is close to that 
of seawater, about 10-12 mM, some fourfold higher 
than in the blood of vertebrates (325). 
Information on osmoregulatory mechanisms and the 
routes of salt excretion is lacking in spiders, with the 
exception of the mygalomorph Porrhothele antipodi- 
ana. Mygalomorphs are considered a primitive group 
and include the tarantulas and trapdoor spiders. Both 
the gut-derived anal system and the coelomoduct- 
derived coxal organs contribute to ionoregulation in 
1222 HANDBOOK OF PHYSIOLOGY-COMPARATIVE PHYSIOLOGY 
P. antipodianu (53). The anal system consists of a pair 
of Malpighian tubules, the midgut diverticula, and the 
stercoral pocket, a structure analogous to the rectum 
in insects. The Malpighian tubules branch from the 
midgut, just anterior to the stercoral pocket, and are 
not homologous, therefore, with insect and myriapod 
tubules, which arise from the hindgut and are thus of 
ectodermal origin. Although they branch dichoto- 
mously within the abdominal mass of storage tissue 
and midgut diverticula, they are surrounded by fat cells 
and are not closely associated with the diverticula. 
Overall, the functioning of this system shows many 
similarities to the Malpighian tubulehindgut system of 
insects, though the means by which hemolymph is 
supplied to the tubules is problematic since they do 
not lie free in the hemocoel. It is worth noting that the 
Malpighian tubules are not involved in K + excretion 
from the anus in another group of chelicerates, the 
ixodid ticks (211). The tick midgut is relatively imper- 
meable to K + , and K + ingested in the blood meal 
probably passes directly from midgut to rectal sac. In 
contrast, the anal system of P. antipodianu is responsi- 
ble for most of the K + excretion (86%) after feeding 
on cockroach nymphs. Anal diuresis lasts for about 3 
days following a single meal, and urine production 
increases sevenfold relative to the rate in starved ani- 
mals. The urine of fed spiders is K+-rich (Na+/ 
K+=0.2) relative to that of starved animals (Na+/ 
K + = l ) . Elevation of the N a + or K + content of the 
prey increases the content of the corresponding ion in 
the anal fluid and the rate of fluid excretion from 
the anus. 
More than two-thirds of the total Na+ excretion 
after a meal is accomplished by the coxal organs, which 
transfer Na+ back into the prey remains, increasing 
the Na+/K+ ratio of the remains from 0.47 to 0.96. 
The coxal organs resemble the filtration-type excretory 
organs of mites, argasid ticks, and crustaceans. The 
term coxal “organ” is preferred, therefore, over the 
more common coxal “gland” (54) because it does 
not imply either the mechanism of fluid production 
(filtration vs. cellular transport) or the function of the 
fluid (secretion vs. excretion). Coxal fluid is produced 
continuously during feeding in P. antipodianu and 
transported from the openings of the coxal organs to 
the preoral region via a hydrophilic cuticular groove 
on the ventral surface of the cephalothorax. Inulin 
injected into the hemocoel appears in the coxal fluid, 
suggesting that the fluid is formed by ultrafiltration. 
Moreover, the coxal fluid is hypoosmotic to the hemo- 
lymph by about two-thirds, and its Na/K ratio is much 
lower than that of the hemolymph (3.8 vs. 44). These 
results suggest secondary secretion of K + and reabsorp- 
tion of Na+. Elevation of the Na+ content of the prey 
or injection of Na+-rich saline into the spider increases 
the Na+ content and rate of excretion of coxal fluid, 
probably as a result of decreased reabsorption of Na+. 
K + loading of prey does not alter ion content or fluid 
excretion rate. 
Although reduction of body volume is the main 
function of diuresis in blood-feeders, in spiders its main 
function is elimination of excess ions ingested with the 
meal. In P. antipodianu, the volume increase is only 
11%, and the spider must actually drink to support 
the prolonged diuresis because much water is lost by 
transpiration. A large volume of fluid is required be- 
cause Na+, K+, and CI- contribute less than 50% of 
the osmotic pressure of the blood and the urine is 
about 2% hypoosmotic compared to the hemolymph 
(515 mOsm. kg-’). High concentrations of guanine 
are present in stercoral fluid, but since this compound 
has a low solubility, it is unlikely to contribute much 
to urine osmotic pressure (53). 
In addition to their roles in ionoregulation, both anal 
and coxal excretory systems have important ancillary 
functions. As noted above, anal urine is the route 
of nitrogenous waste excretion and the coxal organs 
function in prey ingestion. Appearance of the dye ama- 
ranth in the gut lumen after application to the coxal 
openings suggests that much of the coxal fluid is recy- 
cled through the gut. Delivery of coxal fluid to the 
preoral region may facilitate the intake of predigested 
prey by the sucking stomach, perhaps preventing a rise 
in viscosity of the meal by evaporative concentration 
(54). The coxal organ has been reduced and simplified 
in advanced web-spinning spiders,