Integrative and Comparative Biology
Integrative and Comparative Biology, volume 55, number 5, pp. 816–829
doi:10.1093/icb/icv013
Society for Integrative and Comparative Biology
SYMPOSIUM
Links between Osmoregulation and Nitrogen-Excretion in Insects
and Crustaceans
Dirk Weihrauch* and Michael J. O’Donnell1,†
*Department of Biological Science, University of Manitoba, 50 Sifton Road, Winnipeg, Manitoba, Canada R3T 2N2;
†
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1
From the symposium ‘‘Linking Insects with Crustacea: Comparative Physiology of the Pancrustacea’’ presented at the
annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2015 at West Palm Beach, Florida.
1
E-mail: [email protected]
Synopsis The epithelia involved in ionoregulation and detoxification in crustaceans and insects are quite distinct: the
gills, hepatopancreas, and antennal gland serve these functions in crustaceans, whereas the Malpighian tubules, hindgut,
and, to some extent, the midgut, are involved in insects. This article compares the means by which the Naþ/Kþ-ATPase
and the vacuolar type Hþ-ATPase are used to energize ionoregulatory processes in both groups. The vacuolar Hþ-ATPase
is particularly important as a generator of both Hþ gradients and transmembrane electrical gradients that can be used to
energize electroneutral or electrogenic exchange of Naþ and/or Kþ for Hþ. In addition to cation:proton antiporters,
epithelia in both groups depend upon the activity of Naþ:Kþ:2Cl cotransporters, Cl/HCO3 - exchangers, and channels
for Kþ and Cl for transepithelial ion transport. This article also contrasts the dominant role of ammonia as the primary
nitrogenous waste in crustaceans, with the excretion of ammonia, uric acid, or both in insects.
Epithelial ion transporters in insects and
crustaceans: the major players
The Naþ/Kþ-ATPase
The Naþ/Kþ-ATPase is composed of three subunits
(a, b, and g) of which the a-subunit exhibits the
catalytic function, hydrolyzing ATP (Therien and
Blostein 2000), and presents the binding site for ouabain, a somewhat specific inhibitor of this pump.
The Naþ/Kþ-ATPase usually is localized to the basolateral membrane of epithelial cells and promotes the
transport of 3 Naþ out of the cytoplasm in exchange
for 2 Kþ into the cytoplasm, thereby generating low
intracellular [Naþ] and also contributing to an
inside-negative potential of the cell membrane.
In crustaceans, this pump is key for energizing
osmoregulatory processes and is highly abundant in
the NaCl-transporting tissues, e.g., gills, pleopods,
antennal glands, maxillary glands, and recently described Crusalis organs (Siebers et al. 1982; DeVries
et al. 1994; Postel et al. 2000; Towle and Weihrauch
2001; Henry et al. 2012; Johnson et al. 2014).
In insects, the Naþ/Kþ-ATPase typically plays a
subordinate role in osmoregulatory processes, likely
due to insects’ terrestrial life-style and often Naþpoor diet. For instance, in the midgut and
Malpighian tubules of the tobacco hornworm,
Manduca sexta, the pump was not detectable by
high-cycle PCR, with only little expression found in
the hindgut (Weihrauch 2006). However, in the
yellow-fever mosquito, Aedes aegypti, the Naþ/KþATPase was detected in the Malpighian tubules.
Interestingly, whereas the pump was detected only
in the stellate cells of the distal secretory segment
of the tubule, it was also detected in the principal
cells of the proximal reabsorbing segment (Patrick
et al. 2006). In Drosophila, the Naþ/Kþ-ATPase is
located on the basolateral membrane of the
Malpighian tubules (Torrie et al. 2004). In the
locust rectum, Naþ/Kþ-ATPase in the basolateral
membranes appears to be involved in ion recycling
in the lateral intercellular channels between the rectal
cells, resulting in production of a hypo-osmotic
Advanced Access publication April 17, 2015
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Osmoregulation and nitrogen-excretion
absorbate from the rectal contents, which thereby
can become very hyper-osmotic (4three-fold) to
the hemolymph (Phillips and Audsley 1995).
In addition to its importance in osmoregulation,
the Naþ/Kþ-ATPase also plays a critical role in epithelial transport of ammonia in crustaceans (if
not stated otherwise ammonia refers to either NH3
þ
þ
or NHþ
4 ) since it accepts not only K but also NH4
as a substrate (Skou 1960; Mallery 1983). Moreover,
in gills of the portunid crab, Callinectes danae, and
the freshwater shrimp, Macrobrachium olfersii, the
branchial Naþ/Kþ-ATPase is stimulated synergistiþ
cally by NHþ
(Masui et al. 2002; Furriel
4 and K
et al. 2004). A direct involvement of the Naþ/KþATPase in transepithelial ammonia transport has
been shown for the gills of the green crab, Carcinus
maenas, and the edible crab, Cancer pagurus, in
which application of ouabain drastically inhibited
branchial excretion of ammonia (Weihrauch et al.
1998, 1999). For insects, participation of the Naþ/
Kþ-ATPase has not been shown directly, but excretion of ammonia observed in the anal papillae of
aquatic mosquito larvae (Donini and O’Donnell
2005; Larsen et al. 2014) may involve transport of
ammonia from the hemolymph into the cell cytoplasm via the basolateral Naþ/Kþ-ATPase (Patrick
et al. 2006).
The vacuolar Hþ-ATPase
The vacuolar Hþ-ATPase (V-ATPase) is a holoenzyme composed of two complexes, the cytoplasmatic
V1 complex comprised of eight different subunits
(A, B, C, D, E, F, G, and H) and the membrane
integral V0 complex comprised of at least four different subunits (a, c, d, and e). The pump mediates
the translocation of protons out of the cytoplasm
into extracellular fluids (environmental or body
fluids) or into intracellular vesicles and endosomes
(Wieczorek et al. 2009) and can rather specifically be
inhibited by bafilomycin A and by concanamycin
(Bowman et al. 1988, 2004). Central to the operation
of the Hþ-ATPase in osmoregulatory processes is its
electrogenic nature; the voltage gradient created by
ATP-dependent Hþ-transport is coupled to movements of Naþ, Kþ, and Cl through pathways or
transporters that mediate voltage-driven transport.
In crustaceans this is crucial for branchial osmoregulatory uptake of ions in NaCl-poor freshwater
environments, where V-ATPase supports the action
of the Naþ/Kþ-ATPase (Henry et al. 2012). Protonpump expression is abundant in the apical membrane of the gills’ epithelia, as seen in the true
freshwater crab, Dilocarcinus pagei (Weihrauch
817
et al. 2004a) and freshwater-acclimated Chinese
mitten crabs, Eriocheir sinensis (Tsai and Lin 2007).
Interestingly, an up-regulation of V-ATPase gene
expression in the osmoregulatory gills was also
observed in the hypo-regulating crabs, E. sinensis
(Weihrauch et al. 2001) and Chasmagnathus granulatus (Luquet et al. 2005), indicating an important
role for this pump in ion-secretion processes as well.
The V-ATPase is also present in the apical membranes of cells in the antennal gland in terrestrial
crabs (Tsai and Lin 2014). The V-ATPase works in
partnership with carbonic anhydrase, which acts by
hydration of carbonic dioxide to produce both Hþ
and HCO3 - for transport by the V-ATPase and Cl/
HCO3 - exchangers, respectively.
In insects the V-ATPase is of particular importance and can be found in the plasma membranes
of cells in the salivary glands (Baumann and Walz
2012) and in virtually all osmoregulatory tissues, including midgut, hindgut/rectum, Malpighian tubules,
and, in mosquito larvae, the anal papillae (Dow et al.
1997; Patrick et al. 2006; Harvey 2009; Onken and
Moffett 2009; Wieczorek et al. 2009; Blaesse et al.
2010; Beyenbach and Piermarini 2011).
In contrast to the Naþ/Kþ-ATPase, the V-ATPase
cannot directly transport ammonia, but the protontransporting feature of this pump plays a key role in
transepithelial excretion of ammonia and in many
uptake-processes investigated to date (Larsen et al.
2014). Diffusion across membranes via NH3-channels
(Rh-proteins, see below) strictly depends on the
transmembrane partial pressure-gradient of NH3
(PNH3), most often mediated by the proton-pumping action of the V-ATPase. The resulting local acidification of one side of the membrane causes the
conversion of existing NH3 (pKa 9.3) to NHþ
4,
thereby generating a transmembrane PNH3 through
a mechanism known as ‘‘ammonia trapping’’.
Other transporters involved in osmoregulation and in
excretion of ammonia
Members of the monovalent cation proton antiporter
(CPA) superfamily have been implicated in important roles in osmoregulation and in excretion of ammonia in insects and crustaceans. In eukaryotic
organisms the superfamily can be subdivided into
CPA1 (cation/proton exchangers; NHEs) and CPA2
(cation/proton antiporters; NHAs) (Brett et al. 2002;
Rheault et al. 2007). In contrast to the electroneutral
NHEs described in vertebrate species, NHEs in invertebrates are thought to be electrogenic, transporting two cations (Kþ or Naþ, possibly NHþ
4 ) in
exchange for one proton, driven by a negative cell
818
potential and/or a low cellular concentration of Kþ
or Naþ (Shetlar and Towle 1989; Ahearn et al. 2001;
Piermarini et al. 2009; Blaesse et al. 2010). So far,
molecular biological studies on crustaceans have revealed only one NHE, which is expressed predominantly in the gills (Towle et al. 1997) and also in the
apical membrane of the labyrinthine cells and endlabyrinthine cells of the antennal glands in semiterrestrial crabs (Tsai and Lin 2014). Experiments
employing brush-border membrane (BBM)-vesicles
from lobster, however, point strongly toward the existence of further NHEs localized in the antennal
gland and in the hepatopancreas, respectively,
accepting either 2 Naþ or 1 Ca2þ as a substrate for
exchange with Hþ (Ahearn et al. 2001). For insects,
three NHEs have been described to date and have
been partially characterized. The Drosophila members
of the NHE family (DmNHE1, DmNHE2, and
DmNHE3) are widely expressed in head, body, and
Malpighian tubules (Giannakou and Dow 2001). The
nomenclature for different species of insect is confusing and in the following NHEs will be named
according to their clustering with the mammalian
NHEs, with the corresponding nomenclature from
Drosophila provided in brackets. Studies on the
yellow-fever mosquito, A. aegypti, revealed that
AeNHE3 (DmNHE2) is expressed predominantly in
the basolateral plasma membrane of the Malpighian
tubules, in the midgut, and in the ion-transporting
sector of the gastric caeca (Pullikuth et al. 2006). The
AeNHE8 (DmNHE1) isoform, which is sensitive
to ethyl isopropyl amiloride (EIPA), was found in
Malpighian tubules, but in intracellular compartments rather than in the plasma membrane
(Piermarini et al. 2009). An analysis of mRNA expression in M. sexta revealed further that MsNHH8
is rather evenly distributed among tissues, suggesting
a housekeeping function for this transporter (Blaesse
et al. 2010). In contrast to MsNHE8, MsNHE7, 9
(DmNHE3) showed higher levels of expression in
the hindgut and Malpighian tubules of M. sexta
than in the anterior or posterior midgut (Blaesse
et al. 2010). There is evidence from the midgut of
M. sexta that the uptake of ammonia is sensitive to
EIPA and amiloride and that uptake is electroneutral
and likely involves an apically localized NHE
(Weihrauch 2006; Blaesse et al. 2010).
For members of the CPA2 subfamily only limited
information is available. These transporters share sequence-homology to prokaryote NHAs (NhaA,
NhaB), which promote an electrogenic Naþ/nHþ exchange, driven by differences in voltage and [Hþ]
(Padan et al. 2001). Although no NHAs have been
identified in crustaceans, there are two isoforms in
D. Weihrauch and M. J. O’Donnell
insects, with high levels of expression found in the
gastric caeca and rectum in mosquitoes. According
to their clustering with their prokaryotic homologs,
an electrogenic Naþ/nHþ transport modus was also
suggested for insects’ NHAs (Rheault et al. 2007). In
Drosophila, two members of the CPA2 gene family
co-localize to the same apical membrane of the
Malpighian tubule as the plasma-membrane
V-ATPase, and also show distinct specificities for
Naþ versus Kþ (Day et al. 2008).
Other transporters usually involved in osmoregulatory transport in insects and crustaceans include
Kþ channels, Naþ/Kþ/2Cl cotransporters, Naþ
channels, Cl/HCO3 - exchangers, and Cl channels.
Kþ channels often, if not always, are co-localized
with the Naþ/Kþ-ATPase and provide for recycling
of Kþ ions and for maintenance of the membrane
potential (Freire et al. 2008). Pharmacological experiments in the green crab, C. maenas, have implicated
these channels in branchial excretion of ammonia as
well (Weihrauch et al. 1998), supported by the fact
that Kþ channels, in general, allow NHþ
4 transport to
a certain extent (Choe et al. 2000). In insects, Kþ
channels also play a crucial role in osmoregulatory
processes, mediating secretion of Kþ that is driven by
the Naþ/Kþ-ATPase or V-ATPase in the Malpighian
tubules (Beyenbach et al. 2010).
Except in epithelia exposed to freshwater, the
Naþ/Kþ/2Cl cotransporter plays a fundamental
role in osmoregulatory transport of ions and can
be found in gills and antennal glands of crustaceans
and in Malpighian tubules of insects. Depending on
the direction of ion transport this electroneutral
transporter is localized to the basolateral or apical
plasma membrane, respectively, for the secretion or
uptake of ions. An intriguing aspect of cotransporter
function in the Malpighian tubules of Rhodnius
prolixus is that Naþ can substitute for Kþ; the
cotransporter may thus operate as a 2Naþ:2Cl
cotransporter, and it plays an important role in the
secretion of excess Naþ into the lumen of the tubule
during post-prandial diuresis (Ianowski et al. 2004).
Cl channels in epithelial cells are localized to the
membrane opposite that of the Naþ/Kþ/2Cl cotransporter, and are responsible for the cellular exit
of Cl, driven by the electrochemical gradient
(Tse et al. 1993; Beyenbach et al. 2010; Henry et al.
2012). Usually localized to the same plasma membrane as the Naþ/Kþ/2Cl cotransporter is the electroneutral HCO3 -/Cl exchanger (anion exchanger,
AE). This transporter is driven by intracellularto-extracellular gradients in HCO3 - and promotes
an uptake of Cl into the cytoplasm. In gills of
freshwater crustaceans AE may be the dominant
Osmoregulation and nitrogen-excretion
transporter for uptake of Cl from the environment
across the apical membrane (Henry et al. 2012).
However, high levels of mRNA-expression also have
been found in the osmoregulatory gills of C. maenas
(Fehsenfeld and Weihrauch 2012). To our knowledge, Naþ channels have been implicated in osmoregulatory processes only in gills of freshwater
crustaceans, mediating Naþ uptake from the environment. This uptake is driven by a combination
of low intracellular [Naþ] and a strongly hyperpolarized membrane potential (Henry et al. 2012).
While some transporters involved in osmoregulation (e.g., Naþ/Kþ-ATPase or Kþ channels) may also
transport ammonium (NHþ
4 ), Rhesus-like proteins
are NH3 channels (Gruswitz et al. 2010) and thus
require a partial pressure gradient for NH3
(PNH3). In crustaceans, only one Rh-isoform has
been identified to date and it is localized predominantly in ammonia-excreting gills (Weihrauch et al.
2004b; Martin et al. 2011). Insects (mosquito Aedes
sp.) express two different isoforms of Rh-proteins,
named Rh50-1 (alternatively Rhb) and Rh50-2
(alternatively Rha). For both isoforms the capability
for transporting ammonia was confirmed when functionally expressed in yeast (Pitts et al. 2014). While
Rh50-1 showed high levels of expression in the antenna, and possibly serves as an ammonia sensor
(Pitts et al. 2014), high abundance for Rh50-2 was
detected in the ganglia, hindgut, and Malpighian
tubules of the tobacco hornworm (Weihrauch
2006) and in the ammonia-transporting anal papillae
of mosquito larvae (Weihrauch et al. 2011). While
Rh-proteins are highly expressed in the Malpighian
tubules and in the hindgut of M. sexta (Weihrauch
2006) and in Drosophila melanogaster (FlyAtlas
(Chintapalli et al. 2007)), the presence of these transporters was also verified in the anal papillae of mosquito larvae (Weihrauch et al. 2011). However, for
neither crustaceans nor insects is the subcellular
localization of these NH3-channels known.
Organs and tissues involved in
osmoregulation
Crustaceans
Most studies of osmoregulation and its underlying
mechanisms in crustaceans have been conducted on
decapod crabs, in which the most important organs
for osmoregulatory processes are the posterior gills.
These gills typically exhibit a high abundance and
activity of the Naþ/Kþ-ATPase, which is essential
for all branchial osmoregulatory processes (Siebers
et al. 1982; Towle and Weihrauch 2001; Henry
et al. 2012). The uptake of NaCl in gills of weak
819
osmoregulators, such as the green crab, C. maenas,
is mediated by the basolateral Naþ/Kþ-ATPase,
which drives, due to the generation of a low cytoplasmatic [Naþ], an apical Naþ/Kþ/2Cl cotransporter (Henry et al. 2012). In addition, high rates of
activity of carbonic anhydrase further suggest an
apical uptake of Naþ and Cl via putative apical
NHE and AE, respectively, which are also highly expressed under hyper-regulatory conditions (Towle
et al. 1997, 2011). Whereas Cl is transported basolaterally via Cl channels, the majority of transepithelial uptake of Naþ is thought to be driven by
the negative transepithelial difference in potential via
the paracellular pathway across this rather leaky epithelium (Gte ca. 45 mS cm2 [Riestenpatt et al.
1996]) (Fig. 1). A very similar mechanism for the
uptake of NaCl also was suggested for the posterior
endopodites of the hyper-regulating isopod, Idotea
baltica (Postel et al. 2000).
There are only a few studies addressing the mechanisms of NaCl excretion across the branchiae of
hypo-regulating crustaceans. Research on the semiterrestrial South American crab, C. granulatus, revealed that the levels of mRNA-expression of Naþ/
Kþ-ATPase, V-ATPase, and Naþ/Kþ/2Cl are upregulated in NaCl-excreting, posterior gills during
hypo-regulation, relative to non-ionoregulatory conditions when crabs are acclimated to 30ø seawater
(Luquet et al. 2002, 2005). Also, in the gills of the
hypo-regulating Chinese mitten crab, E. sinensis,
an increase in gene expression of V-ATPase
(Weihrauch et al. 2001) and Naþ/Kþ/2Cl cotransporter (D. Weihrauch, unpublished data) was detected. The presence and up-regulation of an Naþ/
Kþ/2Cl cotransporter in hypo-regulating gills
suggests a mechanism of excreting NaCl similar
to that in the proposed model described for the
gills of marine teleost fish (Evans 2008) and the
rectal glands of elasmobranchs (Riordan et al.
1994). In the latter tissues, the Naþ/Kþ/2Cl
cotransporter is basolaterally localized, with Cl
exiting via apical Cl channels. Naþ is thought
to be excreted via the paracellular route. In addition, participation of a V-ATPase is suggested to
play a role in branchial secretion of NaCl in hyporegulating crabs. Although speculative, we suggest
that hyperpolarization of the apical membrane of
the gill’s epithelium by the V-ATPase may facilitate the apical exit of Cl. The overall mechanism
of NaCl excretion in gills of hypo-regulating crustaceans is, however, still unclear and awaits further investigation.
Naþ is not taken up via the paracellular pathway
in the low-conductance gills of freshwater-inhabiting
820
D. Weihrauch and M. J. O’Donnell
Fig. 1 Working model for the branchial uptake of NaCl in the hyper-regulating green shore crab, Carcinus maenas. For detailed
explanation refer to the text. CA, carbonic anhydrase; AE, anion exchanger; NHE, Naþ/Hþ exchanger (CPA1 subfamily).
crabs such as E. sinensis (Gte ca. 3–4 mS cm2
[Weihrauch 1999]) or the true freshwater red crab,
D. pagei (Gte of distal side of gill lamella ca. 4 mS
cm2; Gte of proximal side of gill lamella ca. 18 mS
cm2) (Onken and McNamara 2002). Instead, entry
of Naþ through apical Naþ channels is driven by the
action of a basolateral Naþ/Kþ-ATPase and a
strongly hyperpolarized epithelial cell. Uptake of
Cl on the other hand, is mediated by the concerted
action of an apical V-ATPase and a parallel AE, with
a functioning carbonic anhydrase required to readily
supply protons and bicarbonate (Henry et al. 2012).
Interestingly, in gills of freshwater crabs, Naþ and
Cl seem to be taken up by different cells. In the
branchial epithelium of E. sinensis, the V-ATPase was
localized to the pillar cells (Freire et al. 2008) and in
the gills of D. pagei uptake of Naþ and Cl even
occurs on opposite sides of the gill lamella. While
the distal side features a thin epithelium and mediates the V-ATPase-driven uptake of Cl, the proximal side of the lamella is rather thick and rich
in mitochondria and promotes Naþ/Kþ-ATPasedriven Naþ-uptake (Onken and McNamara 2002;
Weihrauch et al. 2004a). A hypothetical model of
the uptake of NaCl in freshwater crabs is provided
in Fig. 2.
In crustaceans not bearing classical gills, other osmoregulatory organs have been identified, or at least
suggested. Among them are recently described
Crusalis organs, found in the swimming legs in freshwater-invading copepods, Eurytemora affinis. As in the
gills of freshwater brachyurans, high levels of expression of Naþ/Kþ-ATPase and V-ATPase are also found
in the Crusalis organ (Johnson et al. 2014). In hyporegulating brine shrimps, Artemia salina, ion-secretion
is mediated by the metepipodites of the phyllopodia,
which show high conductivity to Cl and a high
activity of Naþ/Kþ-ATPase (Holliday et al. 1990).
In addition to osmoregulatory tissues directly
facing the environment, internal organs are also involved in extracellular volume regulation and in
excretion, namely the antennal and maxillary glands,
respectively. The glands feature a mammalian nephron-like ultrafiltration with modifications of ioncomposition of the primary filtrate in the downstream
sections of the gland (Freire et al. 2008; Tsai and Lin
2014). In the crayfish, Pacifastacus sp. and Orconectes
virilis, molecules of a molecular mass less than 20 kDa
are filtered freely into coelomic end-sac, the first part
of the gland. The antennal glands of some, but not all,
crustaceans inhabiting freshwater or water of low salinities are able to produce a dilute urine (Mantel and
821
Osmoregulation and nitrogen-excretion
Farmer 1983; Santos and Salomao 1985; Freire et al.
2008). Antennal glands of intermolt crayfish, for example, are capable of reabsorbing approximately 90%
of the filtrated ions due to the elevated activities of
carbonic anhydrase, Naþ/Kþ-ATPase, and Ca2þATPase (Freire et al. 2008). A study on the semi-terrestrial crab, Ocypode stimpsoni, showed that three
major cell-types are present in the antennal gland:
(1) labyrinthine cells; (2) end-labyrinthine cells; and
(3) coelomic cells. The labyrinthine cells are found
predominantly in the proximal tubular region of the
antennal gland, with decreasing abundance in the
distal and end regions. These cells exhibit a classical
BBM and basolateral infoldings with associated mitochondria. Immunohistochemistry revealed the presence of a basolateral Naþ/Kþ-ATPase and Naþ/Kþ/
2Cl cotransporter, as well as an apical V-ATPase and
NHE (Tsai and Lin 2014). The basolateral location of the
Naþ/Kþ/2Cl cotransporter suggests secretion of NaCl
as a function of the cell, while the BBM also implies
reabsorption, likely of sugars and amino acids. The
end-labyrinthine cells are found predominantly in the
end-tubular region. This cell-type exhibits an irregular
apical region and multiple basolateral infoldings with
mitochondria distributed throughout the cytoplasm.
While the Naþ/Kþ-ATPase is basolaterally localized in
these cells, V-ATPase, NHE, and Naþ/Kþ/2Cl cotransporter are found on the apical membrane, suggesting a
function in NaCl-uptake. Additionally, Naþ/KþATPase activity/abundance increases from the proximal
tubular region toward the distal/end tubular region, as
also observed in crayfish and lobsters (Peterson and
Loizzi 1974; Khodabandeh et al. 2005; Tsai and
Lin 2014). The coelomic cells are found in similar
regions as are the labyrinthine cells and are of similar
abundance. However, coelomic cells are likely not
involved in osmoregulation (Tsai and Lin 2014).
While the organization of the antennal gland seems to
be very similar in many crustaceans, the ability to
produce a diluted urine was found so far only in species
inhabiting very dilute or freshwater environments
(Freire et al. 2008).
Insects
Whereas crustaceans are aquatic and the primary
inorganic cation regulated by their transporting epithelia is Naþ, most insects are terrestrial and the
plant-based diet is rich in Kþ and low in Naþ. As
a consequence, mechanisms for retention of water
are paramount and the transport of inorganic cations
by the gut and Malpighian tubules is concerned primarily with Kþ rather than with Naþ. Blood-feeders
Fig. 2 Working model for the branchial uptake of NaCl in freshwater crabs exhibiting two distinct cell-types. For detailed explanation
refer to the text. CA, carbonic anhydrase; AE, anion exchanger; GJ, gap junction.
822
such as mosquitoes and triatomid bugs are exposed
to high loads of Naþ immediately following the
meal, but must later deal with Kþ released as the
erythrocytes are digested, and so potent mechanisms
for elimination of both cations are required.
Mechanisms of retention of water include an impermeable epicuticle (Gibbs 2011; Gibbs and Rajpurohit
2010), mechanisms for closing the spiracles, and discontinuous ventilation (White et al. 2007), as well as
recovery of water in the hindgut (Phillips et al. 1986;
Coast et al. 2002; Larsen et al. 2014).
The primary osmoregulatory and excretory organs
in insects are the Malpighian tubules and the hindgut, although the midgut may play an ancillary role.
For example, organic anions and cations may be excreted across the midgut (Bijelic et al. 2005; RuizSanchez and O’Donnell 2007), which is richly
D. Weihrauch and M. J. O’Donnell
endowed with toxin-transporters such as P-glycoproteins in species such as D. melanogaster (Tapadia and
Lakhotia 2005). Together, the Malpighian tubules
and hindgut form the functional kidney; primary
urine secreted by the tubule is modified by secretory
and reabsorptive processes downstream in a segment
of the tubule close to the junction with the hindgut
or, more commonly, in the hindgut itself (O’Donnell
2008). Whereas production of the primary urine is a
consequence of ultrafiltration driven by blood pressure in the vertebrate kidney, secretion of fluid by
the Malpighian tubule is an osmotic consequence of
the active transport of ions, primarily Kþ, Naþ, and
Cl, into the lumen of the tubule. Mechanisms of
secretion and reabsorption of inorganic ions in the
Malpighian tubules of the hematophagous insect
R. prolixus are summarized in Fig. 3A–C. The
Fig. 3 Working models for transport of inorganic ions by the Malpighian tubules of the hematophage, R. prolixus (A–C) and the fruit fly
D. melanogaster (D). (A). Naþ, Kþ, Cl, and H2O are secreted by a single cell-type in the upper Malpighian tubule (UMT) of R. prolixus.
Kþ and Cl are reabsorbed by the lower Malpighian tubule (LMT). Although Kþ and Cl are reabsorbed in the rectum in many insect
species, the higher surface-area to volume ratio of the LMT presumably provides for more effective reabsorption during high rates of
the flow of fluid achieved during the diuresis after ingestion of blood. (B) Ion transporters in the UMT of R. prolixus. Transport of Hþ
into the tubule’s lumen by the V-ATPase establishes the proton motive force to drive Naþ and Kþ into the lumen through Naþ(Kþ)/Hþ
exchangers. The V-ATPase also creates a positive apical membrane-potential in the lumen, thereby providing a favorable gradient for
the movement of Cl from cell to lumen. Entry of Kþ, Naþ, and Cl through a basolateral Naþ:Kþ:2Cl cotransporter utilizes the Naþ
gradient created by the apical exchangers (Ianowski et al. 2002; Ianowski and O’Donnell 2006). (C) Ion transporters in the LMT of
R. prolixus. Reabsorption of Kþ and Cl is proposed to involve an omeprazole-sensitive Hþ/Kþ-ATPase and Cl/HCO3 - exchangers,
respectively. Exit of Kþ and Cl from cell to lumen is via conductive pathways (i.e., channels) (Haley and O’Donnell 1997; Haley et al.
1997). (D) Ion transporters in the Malpighian tubules of D. melanogaster. The tubules are comprised of principal cells and stellate cells.
The V-ATPase creates an electrical gradient for the operation of a CPA2 exchanger (Day et al. 2008) and also establishes the positive
transepithelial potential that drives Cl from cell to lumen through ClC-a Cl channels in the stellate cells (Cabrero et al. 2014)
and possibly through additional, uncharacterized Cl channels in the apical membrane of the principal cells (Miller et al. 2013). Entry
of ions into the cells across the basolateral membrane involves Naþ:Kþ:2Cl cotransporters (Ianowski and O’Donnell 2004), Naþdependent Cl/HCO3 - exchange (Sciortino et al. 2001), and the Naþ/Kþ-ATPase (Linton and O’Donnell 1999; Torrie et al. 2004).
In tubules of A. aegypti, Ba2þ-sensitive Kþ channels (not shown) also are implicated in Kþ transport across the basolateral membrane
(Piermarini et al. 2012).
Osmoregulation and nitrogen-excretion
upper tubule secretes a nearly isosmotic fluid (370
mOsm) containing approximately 75 mM Kþ and
125 mM Naþ. Fluid is modified downstream by reabsorption of Kþ and Cl but not of water in the lower
tubule. The composition of the final urine (125 mM
Naþ, 3 mM Kþ, and 120 mM Cl) is such that much
of the plasma fraction of the blood-meal is eliminated during diuresis and that both hemolymph
Kþ concentration and osmolality are preserved
when feeding on hypos-osmotic avian or mammalian
blood (320 mOsm) (Maddrell and Phillips 1975).
Whereas ion-secretion is accomplished by a single
cell-type in the upper tubule of R. prolixus, tubules
of many species have both principal and stellate cells.
In dipterans such as Drosophila, the stellate cells mediate much of the transepithelial transport of Cl
and water (Fig. 3D).
The fat body, analogous to the crustacean hepatopancreas and the vertebrate liver, is the primary
organ of intermediary metabolism in insects and is
a major source of nitrogenous wastes such as uric
acid (Buckner et al. 1993) and ammonia (Scaraffia
et al. 2005), which are then transferred through the
hemolymph to the Malpighian tubules. The hindgut
is also an important site for elimination of ammonia.
In locusts, for example, the rectum secretes significant quantities of endogenously produced ammonia
preferentially into the lumen as NHþ
4 rather than as
NH3 (Thomson et al. 1988).
The tubules not only provide the primary pathway
for elimination of nitrogenous wastes and organic
toxins, but are themselves important sites of detoxification. For example, ingestion of the insecticidesynergist piperonyl butoxide is associated with
alterations in the expression of multiple genes for
detoxification enzymes, such as the cytochrome
P450s (Phase-I, oxidation or hydrolysis) and glutathione-S-transferases (Phase-II, conjugation) (Chahine
and O’Donnell 2011). Changes in the levels of a
single P450 gene, Cyp6g1, in just the Malpighian
tubules of adult Drosophila, are correlated with
changes in survival after exposure to 1,1,1-trichloro2,2-bis(4-chlorophenyl)ethane (Yang et al. 2007).
Excretion of nitrogen
Crustaceans
With the exception of the terrestrial robber crab,
Birgus latro, which mostly excretes uric acid
(Greenaway and Morris 1989), all crustaceans investigated so far are, regardless of their habitat, ammonotelic, excreting the majority of their nitrogenous
wastes in the form of ammonia (Larsen et al.
2014). In aquatic crabs the main site of excretion is
823
the gill, which facilitates an active net transport of
ammonia even against a two-fold to 10-fold inwardly
directed gradient. Branchial transport of ammonia
has been shown for various haline species, including
marine osmoconformers with a very leaky epithelium
(C. pagurus, Metacarcinus magister), weak osmoregulators (C. maenas), and freshwater species in
which the gills have a low conductance (E. sinensis)
(D. Weihrauch 1999; Martin et al. 2011).
In contrast to osmoregulatory function, both
anterior and posterior gills are equally involved in
excretion of ammonia, with a tendency for higher
excretion rates in the anterior gills in C. maenas
(Martin et al. 2011; Fehsenfeld and Weihrauch
2012). For C. maenas, it is suggested that hemolymph ammonia (NHþ
4 ) enters the branchial epithelium directly via the Naþ/Kþ-ATPase and also via
Kþ channels, with entry by the latter pathway likely
driven by the negative potential of the cell. Ammonia
imported from the hemolymph and/or produced by
cellular metabolism within the gill diffuses as NH3
into acidified vesicles and is trapped in this compartment as NHþ
4 . Participation of an Rh-protein in this
process is suggested on the basis of preliminary results that show a cytoplasmic signal in response to an
Rh-specific antibody (D. Weihrauch, unpublished
data). Ammonium-laden vesicles are then transported via the network of microtubules toward the
apical membrane, where NHþ
4 is released by exocytosis into the subcuticular space (Weihrauch et al.
2004b). In addition, the predicted presence of an
apical NHE suggests an ammonia-trapping mechanism, provided that an Rh-protein is co-localized
to the membrane facilitating transmembrane transport of NH3. Further, a paracellular movement of
NHþ
4 across this rather leaky gill epithelium cannot
be excluded. The hypothetical mechanism of excretion by the gills is illustrated in Fig. 4. Interestingly, a
vesicular mechanism for transporting ammonia was
also predicted to be involved in the active uptake of
ammonia in the midgut of the tobacco hornworm
M. sexta (Weihrauch 2006).
Branchial excretion of ammonia in freshwater
crabs might involve a different, so-far-unknown
mechanism, including the participation of the Naþ/
Kþ-ATPase and probably apical trapping of ammonia. Vesicular transport of ammonia, however, has
not been shown to date, or even suggested for any
freshwater vertebrate or invertebrate (Weihrauch
et al. 2012; Larsen et al. 2014).
In some terrestrial crabs the antennal gland also
seems to be involved in excretion of ammonia. For
instance, the ghost crab, Ocypode quadrata, produces
an acidic (pH 5.4) and ammonia-rich (116 mM)
824
D. Weihrauch and M. J. O’Donnell
Fig. 4 Working model for the branchial excretion of ammonia in the green shore crab, Carcinus maenas. For detailed explanation refer
to the text. CA, carbonic anhydrase; NHE, Naþ/Hþ exchanger (CPA1 subfamily); Rh, Rh-protein; MT, network of microtubules.
urine that is redirected into the branchial chamber.
Branchial transport-processes then alkalinize the
fluid and NH3 partially volatilizes (Weihrauch et al.
2004b).
In Porcellio scaber, a fully terrestrial isopod, NH3 is
volatilized from the surface of the abdomen, possibly
coupled with the process of active absorption of
water vapor (WVA) (Wright and O’Donnell 1992).
The actual mechanism of transport is unknown;
however, excretion likely occurs only when atmospheric humidities allow WVA. Elevated concentrations of ammonia in the hemolymph were measured
only during high humidities, whereas, under conditions unsuitable for WVA, ammonia is stored as glutamine or arginine (Wright et al. 1994, 1996).
Insects
Insects generally are viewed as uricotelic, in keeping
with the Baldwin–Needham hypothesis that terrestrial animals excrete less-soluble nitrogenous excretion-products, such as urea, uric acid, or allantoin, as
a means of conserving water (Baldwin and Needham
1934). Ammonia generally is considered to be the
most toxic, but also the most soluble, of the nitrogenous wastes, and aquatic insect larvae have long
been known to excrete ammonia. More recently,
analysis of ion transport by the anal papillae of
freshwater mosquito larvae has revealed that the papillae are involved not just in uptake of Naþ and
Cl, but also in excretion of Hþ and ammonia
(Donini and O’Donnell 2005). Uptake of Naþ at
the apical membrane occurs through a phenamilsensitive Naþ channel that is driven by a V-type
Hþ-ATPase and uptake of Cl occurs through a
Cl/HCO3 - exchanger. A current model of transport
proposes that carbonic anhydrase activity provides
Hþ and HCO3 - to the V-type Hþ-ATPase and exchanger, respectively (Del Duca et al. 2011).
Outwardly directed Hþ flux, in part established by
a V-type Hþ-ATPase, may drive excretion of ammonia through acid-trapping of NH3 (resulting in a
measurable flux of NHþ
4 ), utilizing Rhesus-like NH3
channels expressed in this tissue (Weihrauch et al.
2011). In fact, when exposed to media containing elevated NH4Cl levels (1 mM), both Hþ-secretion and
ammonia-flux increased significantly (Weihrauch
et al. 2011). Rates of transport of NHþ
4 per unit surface area of the papillae are similar to those of the
locust hindgut, suggesting that the papillae play an
important role in excretion of nitrogenous waste.
In addition to the aquatic insect larvae described
above, ammonia is also excreted by the hindgut
and, to some extent, by the Malpighian tubules, of
several terrestrial species, including adult cockroaches
825
Osmoregulation and nitrogen-excretion
(Mullins and Cochran 1973), larval blowflies (Prusch
1972), and adult mosquitoes (Scaraffia et al. 2005).
Large amounts of ammonium urate also are excreted
by the desert locust, Schistocerca gregaria (Phillips
et al. 1994). Some insects can excrete a range of nitrogenous end-products simultaneously, including
ammonia, urea, uric acid, and allantoin (Cochran
1985), and alterations in diet may shift the relative
abundances of these nitrogenous wastes.
Ammonium in the excreta of locusts is present
primarily as a precipitate, with urate and organic
ions secreted by the Malpighian tubules, indicating
that excretion of ammonium is compatible with conservation of water in this desert locust. In addition
to providing for elimination of additional nitrogen,
ammonium urate is less soluble than uric acid.
However, ammonium concentrations in the excreta
are three-fold higher than those of urates, suggesting
that much of the NHþ
4 is precipitated with organic
anions (Harrison and Phillips 1992). Most of the
NHþ
4 is excreted into the ileum through amiloridesensitive Naþ/NHþ
4 exchange. As a consequence, the
ileum is also a major site of acid-excretion through
Hþ trapped by NH3 as NHþ
4 . The Malpighian tubules of locusts secrete fluids containing as much
as 5 mM ammonia, but the amount excreted by the
tubules is less than 10% of total NHþ
4 excreted.
Up to 90% of the nitrogenous material in the excreta of cockroaches such as Periplaneta is in the
form of ammonia and little or no uric acid is detectable (Mullins and Cochran 1973). It appears that
much of the ammonia is produced by the action of
the gut flora. It has long been known that cockroaches accumulate uric acid in the fat body, especially when the animals are on a protein-rich diet,
and that the deposits of uric acid are then depleted
when the insects are transferred to a low-protein diet
(Cochran 1985). The fat body also contains high
levels of the bacterial symbiont Blattabacterium,
which produces vitamins and metabolizes sulfur
and sulfur-containing amino acids for the host.
The bacterium, in turn, relies upon the host’s tissues
for production of the amino acids Gln, Asp, Pro, and
Gly (Patiño-Navarrete et al. 2014). These amino
acids are produced through a chimeric metabolic
pathway in which enzymes are supplied by both
the host and the endosymbiont. Uric acid is degraded to allantoin, allantoic acid, and then urea,
using the host’s enzymes in the fat body. The urea
is then broken down by urease in the endosymbiont,
and the resulting ammonia is synthesized into glutamine using glutamine synthetase supplied by the
host.
Urate also plays a major ionoregulatory/osmorgulatory role in cockroaches. When Periplaneta americana is dehydrated, urate is stored in the fat body as
salts of Naþ and, to some extent, Kþ and NHþ
4 . The
insect thus has a store of inorganic cations that are
released to maintain levels of ions in the hemolymph
when the insect drinks fresh water to rehydrate
(Mullins 1974).
An additional role for uric acid in insects has been
revealed by studies of the hematophage R. prolixus.
Digestion of hemoglobin leads to production of multiple reactive oxygen species from reactions involving
both the iron and the heme group (Graça-Souza
et al. 2006). Although the Malpighian tubules of
R. prolixus secrete urate at high levels after the
blood-meal, rates of synthesis and excretion are balanced so that high concentrations (up to 5 mM) are
retained in the hemolymph. At these levels, urate
accounts for almost all of the scavenging of free
radicals in the hemolymph (Souza et al. 1997).
Summary
Both in insects and crustaceans the V-ATPase and
the Naþ/Kþ-ATPase play dominant roles in osmoregulation and excretion. The gradients in transmembrane electrical potential and/or ionic activity
created by these primary transporters are harnessed
to drive fluxes of inorganic ions and ammonia
through multiple secondary transporters, including
cation:proton antiporters, Rhesus glycoproteins,
Naþ:Kþ:2Cl cotransporters, and chloride–bicarbonate exchangers. Notwithstanding these similarities,
the exigencies of terrestrial life have led to more dramatic adaptations of epithelial transport in the recovery of ions and water by insects.
Funding
This work was supported by Discovery grants from
the Natural Sciences and Engineering Research
Council (Canada) to [M.J.O. and D.W.].
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