Entomology 3rd edition - C.Gillott - Chapter 18 ppt

NITROGENOUS EXCRETION AND SALT AND WATER BALANCE In males of some species of cockroaches, for example, Blattella germanica, a consid-erable amount of uric acid as much as 5% of the live

is meant the removal of unwanted materials and the retention of those that are useful,

to maintain as nearly as possible the best cellular environment Regulation is a function

of the excretory system and is of great importance in insects because they occupy suchvaried habitats and, therefore, have different regulatory requirements Terrestrial insects losewater by evaporation through the integument and respiratory surfaces and in the process ofnitrogenous waste removal Brackish-water and saltwater forms also lose water as a result ofosmosis across the integument; in addition, they gain salts from the external medium Insectsinhabiting fresh water gain water from and lose salts to the environment The problem ofosmoregulation is complicated by an insect’s need to remove nitrogenous waste products

of metabolism, which in some instances are very toxic This removal uses both salts andwater, one or both of which must be recovered later from the urine

2 Excretory Systems

2.1 Malpighian Tubules—Rectum

The Malpighian tubules and rectum, functioning as a unit, form the major excretorysystem in most insects Details of the rectum are given in Chapter 16, Section 3.4, and onlythe structure of the tubules is described here

The blindly ending tubules, which usually lie freely in the hemocoel, open into thealimentary canal at the junction of the midgut and hindgut (Figure 18.1A) Typically theyenter the gut individually but may fuse first to form a common sac or ureter that leadsinto the gut Their number varies from two to several hundred and does not appear to be

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CHAPTER 18

FIGURE 18.1. (A) Excretory system of Rhodnius Only one Malpighian tubule is drawn in full; (B) junction of proximal and distal segments of a Malpighian tubule of Rhodnius Part of the tubule has been cut away to show the

cellular differentiation; (C, D) sections of the wall of the distal and proximal segments, respectively, of a tubule;

and (E) tip of Malpighian tubule of Apis to show tracheoles and spiral muscles [A, B, E, after V B Wigglesworth,

1965, The Principles of Insect Physiology, 6th ed., Methuen and Co By permission of the author C, D, from

V B Wigglesworth and M M Saltpeter, 1962, Histology of the Malpighian tubules in Rhodnius prolixus Stal.

(Hemiptera), J Insect Physiol 8:299–307 By permission of Pergamon Press Ltd.]

closely related to either the phylogenetic position or the excretory problems of an insect.Malpighian tubules are absent in Collembola, some Diplura, and aphids; in other Diplura,Protura, and Strepsiptera there are papillae at the junction of the midgut and hindgut.With the tubules are associated tracheoles and, usually, muscles (Figure 18.1E) The lattertake the form of a continuous sheath, helical strips, or circular bands and are situatedoutside the basal lamina They enable the tubules to writhe, which ensures that differentparts of the hemolymph are exposed to the tubules and assists in the flow of fluid along thetubules

A tubule is made up of a single layer of epithelial cells, situated on the inner side of abasal lamina (Figure 18.1B–D) In many species where the tubules have only a secretoryfunction (Section 3.2) the histology of the tubules is constant throughout their length and

basically resembles that of the distal part of the tubule of Rhodnius (Figure 18.1C) The

inner (apical) surface of the cells takes the form of a brush border (microvilli) The outer(basal) surface is also extensively folded Both of these features are typical of cells involved

in the transport of materials and serve to increase enormously the surface area across whichtransport can occur Numerous mitochondria occur, especially adjacent to or within thefolded areas, to supply the energy requirements for active transport of certain ions acrossthe tubule wall In many species various types of intracellular crystals occur which are

NITROGENOUS EXCRETION AND SALT AND WATER BALANCE

presumed to represent a form of storage excretion (Section 3.3) Adjacent cells are closely

apposed near their apical and basal margins, though not necessarily elsewhere

In some insects (e.g., Rhodnius), two distinct zones can be seen in the Malpighian

tubule (Figure 18.1C, D) In the distal (secretory) zone the cells possess large numbers of

closely packed microvilli, but very few infoldings of the basal surface Mitochondria are

located near or within the microvilli In the proximal (absorptive) part of the tubule the

cells possess fewer microvilli, yet show more extensive invagination of the basal surface

The mitochondria are correspondingly more evenly distributed In the flies Dacus and

Drosophila, where pairs of Malpighian tubules unite to form a ureter prior to joining the

gut, the ultrastructure of the ureter resembles that of the proximal part of the Rhodnius

tubule, suggesting that the ureter may be a site of resorption of materials from the urine

Yet other species have even more complex Malpighian tubules in which up to four

dis-tinct regions may be distinguished on histological or ultrastructural grounds On the basis

of the structural features of their cells, these regions have been designated as secretory or

absorptive, though it must be emphasized that physiological evidence for these proposed

functions is largely lacking For a survey of insects whose tubules show regional

differen-tiation and a discussion of tubule function in such species, see Jarial and Scudder (1970)

A cryptonephridial arrangement of Malpighian tubules is found in larvae and adults

of many Coleoptera, some larval Hymenoptera and Neuroptera, and nearly all larval

Lepidoptera (Figure 18.2) Here the distal portion of the Malpighian tubules is closely

apposed to the surface of the rectum and enclosed within a perinephric membrane The

sys-tem is particularly well developed in insects living in very dry habitats, and in such species

its function is to improve water resorption from the material in the rectum (Section 4.1)

2.2 Other Excretory Structures

Even in insects that use the rectum as the primary site of osmoregulation, the ileum

may nonetheless be a site for water or ion resorption In other species where the rectum is

unimportant in osmoregulation, serving only to store urine and feces prior to expulsion, the

ileum often takes on this role

In a few insects the labial glands may function as excretory organs In apterygotes that

lack Malpighian tubules the glands can accumulate and eliminate dyes such as ammonia

carmine and indigo carmine from the hemolymph, but there is no evidence that they can

deal similarly with nitrogenous or other wastes The labial glands of saturniid moths excrete

copious amounts of fluid just prior to emergence from the cocoon, and it may well be

that the primary function of the glands is to reduce hemolymph volume and hence body

weight, which, in such large flying insects, needs to be kept as low as possible The midgut

of silkmoth larvae actively removes potassium from the hemolymph, thus protecting the

tissues from the very high concentration of potassium ions present in the leaves eaten by

these insects

In a few insects it appears that the Malpighian tubules, though present, play no part

in nitrogenous excretion In Periplaneta americana, for example, uric acid is not found in

the tubules but does occur in small amounts in the hindgut, which may excrete it directly

from the hemolymph In P americana much uric acid is stored in urate cells in the fat body,

and the major form of excreted nitrogen in this species is ammonia How this reaches the

hindgut lumen in P americana is unclear However, in the flesh fly Sarcophaga bullata,

ammonia, the primary excretory product, is actively secreted as ammonium ions into the

lumen across the anterior hindgut wall

CHAPTER 18

FIGURE 18.2. Cryptonephridial arrangement of Malpighian tubules in Tenebrio larva (A) General appearance.

Note that only three of the six tubules are drawn fully and that in reality the tubules are much more convoluted and have more boursouflures than are shown; (B) cross section through posterior region of cryptonephridial system; (C) details of a leptophragma; and (D) diagram illustrating proposed mode of operation of system Solid arrows indicate movements of potassium, hollow arrows indicate movements of water Numbers indicate osmotic concentration (measured as freezing-point depression) of fluids in different compartments [After A V Grimstone, A M Mullinger, and J A Ramsay, 1968, Further studies on the rectal complex of the mealworm

Tenebrio molitor L (Coleoptera, Tenebrionidae), Philos Trans R Soc Lond Ser B 253:343–382 By permission

of the Royal Society, London, and Professor J A Ramsay.]

NITROGENOUS EXCRETION AND SALT AND WATER BALANCE

In males of some species of cockroaches, for example, Blattella germanica, a

consid-erable amount of uric acid (as much as 5% of the live weight of the insect) is found in the

utriculi majores (part of the accessory reproductive gland complex) The uric acid becomes

part of the wall of the spermatophore and is, in a sense, “excreted” during copulation

3 Nitrogenous Excretion

3.1 The Nature of Nitrogenous Wastes

In nitrogenous wastes structural complexity, toxicity, and solubility go hand in hand

The simplest form of waste (ammonia) is highly toxic and very water-soluble It contains a

high proportion of hydrogen that can be used in production of water It is generally found as

the major excretory product, therefore, only in those insects that have available large amounts

of water, for example, larvae and adults of freshwater species Nonetheless, exceptions are

known, the best examples being the larvae of meat-eating flies and P americana under

certain dietary regimes Generally, however, in insects, as in other terrestrial organisms,

water must be conserved, and more complex nitrogenous wastes are produced, which are

both less toxic and less soluble In the egg and pupal stage the problem is accentuated

because water lost cannot be replaced, and nitrogenous wastes must remain in the body

in the absence of a functional excretory system Most insects, then, excrete their waste

nitrogen as uric acid This is only slightly water-soluble, relatively non-toxic, and contains

a smaller proportion of hydrogen compared with ammonia

However, uric acid is not the only form of nitrogenous waste Usually traces of other

materials (especially the related compounds allantoin and allantoic acid) can be detected,

and in many species one of these has become the predominant excretory product (Bursell,

1967) Urea is rarely a major constituent of insect urine, usually representing less than 10%

of the nitrogen excreted Traces of amino acids can be found in the excreta of many insects,

but their presence should be regarded as accidental loss rather than deliberate excretion

by an insect (Bursell, 1967) Only occasionally has the excretion of particular amino acids

been authenticated; for example, the clothes moth Tineola and the carpet beetle Attagenus

excrete large amounts of the sulfur-containing amino acid cystine Although in tsetse flies

uric acid is the primary excretory product, two amino acids, arginine and histidine, are

important components of the urine These make up about 10% of the protein amino acids

in human-blood; because their nitrogen content is high, it is probably uneconomical to

degrade them, and they are therefore excreted unchanged (Bursell, 1967) The amino acids

voided in honeydew by plant-sucking Hemiptera must be considered as largely fecal and not

metabolic waste products Because of the large amount of water taken in by aphids, it has

been suggested that they might produce ammonia as their nitrogenous waste Indeed, uric

acid, allantoin, and allantoic acid cannot be detected in their excreta However, ammonia

makes up only 0.5% of the total nitrogen excreted, which has led to the suggestion that it

is used (and detoxified) by symbiotic bacteria in mycetomes (Chapter 16, Section 5.1.2)

Table 18.1 contains selected examples to show the variety of nitrogenous wastes produced

by insects

As can be seen in Figure 18.3, uric acid and the other nitrogenous waste products

are derived from two sources, nucleic acids and proteins Degradation of nucleic acids is

of minor importance; most nitrogenous waste comes from protein breakdown followed

by synthesis of hypoxanthine from amino acids The biochemical reactions that lead to

synthesis of this purine appear to be similar to those found in other uric acid-excreting

organisms (Bursell, 1967; Barrett and Friend, 1970)

CHAPTER 18

TABLE 18.1 Nitrogenous Excretory Products of Various lnsectsa ,b

Uric acid Allantoin Allantoic acid Urea Ammonia Amino acids Odonata

Lepidoptera

aFrom Bursell (1967), after various authors.

bThe quantity of nitrogen excreted in the different products is expressed as a proportion of the nitrogen in the predominant end product.

FIGURE 18.3. Metabolic interrelationships of nitrogenous wastes [After E Bursell 1967 The excretion of

nitrogen in insects Adv Insect Physiol 4:33–67 By permission of Academic Press Ltd and the author.]

NITROGENOUS EXCRETION AND SALT AND WATER BALANCE

In addition to the enzymes for uric acid synthesis there are also uricolytic enzymes that

catalyze degradation of this molecule in many insects (Figure 18.3) Uricase has a wide

distribution within the Insecta Active preparations of allantoinase have been obtained from

many species, but the distribution of this enzyme appears to be rather restricted compared

with uricase Although there are reports that indicate the occurrence of allantoicase and

urease in tissue extracts from a few insects, their presence should not be regarded as having

been established unequivocally In other words, when urea and ammonia are produced in

significant amounts, they are probably derived in a manner other than by the degradation

of uric acid The existence of an ornithine cycle for urea production, such as is found in

vertebrates, has not been proved conclusively, even though the constituent molecules of the

cycle (arginine, ornithine, and citrulline) and the enzyme arginase have been identified in

several species (Cochran, 1975) Cochran (1985) suggested that urea is merely a by-product

of the biochemical conversion of arginine to proline, used in flight metabolism (Chapter 14,

Section 3.3.5) Similarly, the way in which ammonia is produced (especially in those insects

in which it is a major excretory molecule) is poorly understood It is generally assumed to

result from deamination of amino acids, but the precise way in which this occurs remains

unclear

It has been suggested that the most primitive state was that in which the complete series

of uricolytic enzymes was present, and ammonia was the excretory material As insects

be-came more independent of water, selection pressures led to loss of the terminal enzymes and

production of more appropriate excretory molecules This simple view should be regarded

with caution Thus, in some caterpillars, diet can affect the nature of the nitrogenous waste

In certain insects substantial quantities of a particular nitrogenous waste molecule are

pro-duced, yet the appropriate enzyme in the uricolytic pathway has not been demonstrated,

and vice versa; that is, the effects of other metabolic pathways may override the uricolytic

system In many insects (especially endopterygotes) the predominant nitrogenous excretory

product changes during development For example, in the mosquito Aedes aegypti urea is

the principal nitrogenous waste in the (aquatic) larvae, while uric acid becomes dominant in

pupae and adult females (von Dungern and Briegel, 2001) In Pieris brassicae (Lepidoptera)

the major excretory product in the pupa and adult is uric acid; in the larva this compound

constitutes only about 20% of the nitrogenous waste, allantoic acid being the predominant

end product (Table 18.1) Indeed, in some Lepidoptera, the ratio of uric acid to allantoin

may fluctuate widely from day to day (Razet, 1961, cited from Bursell, 1967) Of great

interest will be determination of factors that stimulate inhibition or activation (degradation

or synthesis?) of uricolytic enzymes so that the most suitable form of nitrogenous waste is

produced under a given set of conditions

3.2 Physiology of Nitrogenous Excretion

Uric acid is produced in the fat body and/or Malpighian tubules (occasionally the

midgut) and released into the hemolymph How the highly insoluble uric acid is transported

in the hemolymph remains unclear though the most likely means seems to be as the sodium

or potassium salt, or in combination with specific carrier proteins (Cochran, 1985) The uric

acid is secreted into the lumen of the tubules as the sodium or potassium salt, along with

other ions, water, and various low-molecular-weight organic molecules In a typical insect,

for example Dixippus, secretion occurs along the entire length of the tubule No resorption

of materials takes place across the tubule wall, and urate leaves the tubule in solution In the

rectum resorption of water and sodium and potassium ions occurs, and the pH of the fluid

CHAPTER 18

FIGURE 18.4. Movements of water, ions, and organic molecules in the excretory systems of (A) Dixippus and (B) Rhodnius [After R H Stobbart and J Shaw, 1974, Salt and water balance: Excretion, in: The Physiology of

Insecta, 2nd ed., Vol V (M Rockstein, ed.) By permission of Academic Press, Inc and the authors.]

decreases from 6.8–7.5 to 3.5–4.5 The combined effect of water resorption and pH change

is to cause massive precipitation of uric acid Useful organic molecules such as amino acidsand sugars are also resorbed through the rectal wall The Malpighian tubule-rectal wallexcretory system thus shows certain functional analogies with the vertebrate nephron The

excretion of uric acid in Dixippus is summarized in Figure 18.4A.

In Rhodnius, whose tubules show structural differentiation along their length, the cess of excretion is basically the same as in Dixippus However, in Rhodnius only the

pro-distal portion of the tubule is secretory and resorption of water and cations begins in theproximal part Slight change in pH occurs (from 7.2 to 6.6) as the fluid passes along

NITROGENOUS EXCRETION AND SALT AND WATER BALANCE

the tubule and this is sufficient to initiate uric acid precipitation Further water and salt

resorption occurs in the rectum (pH 6.0), causing precipitation of the remaining waste

(Figure 18.4B)

Although allantoin is the major nitrogenous waste in many insects, its mode of excretion

appears to have been studied in only one species, Dysdercus fasciatus (Hemiptera) (Berridge,

1965) This insect is required, because of its diet, to excrete large quantities of unwanted

ions (magnesium, potassium, and phosphate) This, combined with the insect’s inability to

actively resorb water from the rectum, results in the production of a large volume of urine

Because no resorption or acidification occurs which could cause precipitation of uric acid,

this molecule is no longer used as an excretory product Thus, allantoin, which is 10 times

more soluble than uric acid (yet of equally low toxicity), is preferred However, the insect

does not possess a mechanism for actively transporting this molecule from the hemolymph

to tubule lumen; that is, allantoin only moves passively across the wall of the tubule It is

therefore maintained in high concentration in the hemolymph to achieve a sufficient rate of

diffusion into the tubule Whether a similar mechanism occurs in other allantoin-excreting

insects remains to be seen It may be significant that many other allantoin producers are

herbivorous and have the problem of removing large quantities of unwanted ions

The physiological mechanisms for excretion of other nitrogenous wastes are poorly

un-derstood Aquatic insects are presumed to excrete ammonia in very dilute urine, whereas

lar-vae of meat-eating flies such as Lucilia cuprina and S bullata produce highly concentrated,

ammonia-rich excreta, apparently by actively transporting ammonium ions across the

ante-rior hindgut wall Urea probably moves passively into the Malpighian tubules and becomes

concentrated in the hindgut because of its inability to permeate the cuticular lining as water

resorption occurs

3.3 Storage Excretion

An alternative strategy to the removal of wastes through the Malpighian tubule-rectum

system used by some insects is storage excretion, the retention of the wastes in “out of the

way places” within the body In Dysdercus, for example, uric acid is deposited permanently

in the epidermal cells of the abdomen, forming distinct, white transverse bands (Berridge,

1965) Adult Lepidoptera convert much of their waste nitrogen into pteridines that are stored

in the integument, eyes, or wing scales, giving the insects their characteristic color patterns

(Chapter 11, Section 4.3)

At other times storage of urate occurs even when the tubules are working normally

and may be regarded as a supplementary excretory mechanism for occasions when the

tubules cannot cope with all the waste that is being produced In the larval stages of many

species uric acid crystallizes out in ordinary fat body cells and epidermis, even though the

Malpighian tubules are functional It appears that this is caused by the metabolic activity

of the cells themselves (i.e., they are not accumulating uric acid from the hemolymph),

and crystallization occurs by virtue of the particular conditions (pH, ionic content, etc.)

existing in the cells During the later stages of pupation the crystals disappear, the uric

acid apparently having been transferred to the meconium (the collective wastes of pupal

metabolism, released at eclosion) via the excretory system It is worth noting that in many

species the Malpighian tubules are entirely reconstituted during the pupal stage Thus,

storage of uric acid in fat body and epidermal cells is of great importance at this time Yet

other insects, notably termites and cockroaches, retain large quantities of uric acid in special

cells (urocytes) within the fat body However, as Cochran (1985) pointed out, this is not a

in the adult Dyes present in food are often accumulated in fat body cells where they appear

to become associated with particular proteins These proteins are then transferred to the eggduring vitellogenesis and the dyes subsequently “excreted” during oviposition

Nephrocytes (Chapter 17, Section 2) accumulate a variety of substances, especiallypigments, and their name is derived from the mistaken idea that storage excretion is one oftheir major functions As Locke and Russell (1998) pointed out, nephrocytes are involved

in the metabolism of hemolymph macromolecules

4 Salt and Water Balance

Salt and water balance involves more than simply the control of hemolymph osmoticpressure; the relative proportions of the ions that contribute to this pressure must be main-tained within narrow limits The osmotic pressure of the hemolymph is generally withinthe same limits as that of the blood of other organisms, but it can be increased considerablyunder specific conditions (by the addition, for example, of glycerol, which serves as anantifreeze during hibernation) Regulation of the salt and water content is obviously related

to the nature of the external environment Insects in different habitats face different osmoticproblems Nevertheless, these problems have been solved using the same basic mechanism,namely, the production of a “primary excretory fluid” in the Malpighian tubules followed

by differential resorption from or secretion into this fluid when it reaches the rectum Forclarity the problems of insects living on land, in fresh water, or in brackish or salt water areconsidered separately However, considerable similarity in the solution of these problemswill be seen

4.1 Terrestrial Insects

Terrestrial insects appear able to regulate their hemolymph osmotic pressure over a

wide range of conditions For example, in Tenebrio the hemolymph osmotic pressure varies

only from 223 to 365 mM/l (measured as the equivalent of a sodium chloride solution)over a range of relative humidity from 0% to 100% (Marcuzzi, 1956, cited in Stobbart and

Shaw, 1974) In starving Schistocerca there is only a 30% difference in hemolymph osmotic

pressure between animals kept in air at 100% relative humidity and given only tap waterand those kept in air at 70% relative humidity and given saline (osmotic pressure equivalent

to 500 mM/l sodium chloride) to drink (Phillips, 1964a)

In terrestrial insects water is lost (1) by evaporation across the integument, althoughthis is considerably reduced by the presence of the wax layer in the epicuticle (Chapter 11,Section 2); (2) during respiration through the spiracles [many insects possess devices bothphysiological and structural for reducing the loss (Chapter 15, Section 2.2)]; and (3) duringexcretion Despite these adaptations, insects that inhabit extremely dry environments maybecome greatly dehydrated For example, some desert beetles can survive the loss of 75%

of their body water The critical factor for these beetles is to maintain the intracellularwater concentration by using the water in the hemolymph; in other words, the hemolymph

NITROGENOUS EXCRETION AND SALT AND WATER BALANCE

volume is reduced To avoid the potential osmotic problems that this withdrawal of water

creates, osmotically active particles can be excreted or rendered inactive; for example, ions

are chelated and amino acids are polymerized into peptides The strategy used appears to

be correlated with the insects’ diet: carnivorous species, whose food contains abundant

sodium, tend to excrete the excess ions The diet of herbivores, by contrast, is deficient in

sodium, so these species use chelation as a means of retaining the sodium within the body

(Pedersen and Zachariassen, 2002)

The major source of water for most terrestrial insects is obviously food and drink Some

insects may eat excessively solely for the water content of the food Where sufficient water

cannot be obtained by drinking or in food, the insect must obtain it by other means One

source is the water produced during metabolism Absorption of water vapor from the

atmo-sphere is a method employed by a few insects (e.g., Thermobia and Tenebrio) that are

nor-mally found in very dry conditions Interestingly, the site of absorption is the rectum, which,

as is noted below, is the site of uptake of liquid water in other terrestrial and saltwater insects

Small amounts of ions are lost from the body via the excretory system, and these are

readily made up by absorption across the midgut wall Indeed, in terrestrial insects the usual

problem is removal of unwanted ions present in the diet The food often contains ions in

concentrations that are widely different from those of the hemolymph It is probable that

these ions enter the hemolymph passively in the same proportions as they occur in the diet,

and excesses are subsequently expelled via the excretory system In other words, the midgut

does not act as a selectively permeable barrier to the entry of ions (Stobbart and Shaw, 1974)

The role of the Malpighian tubules and rectum was investigated by examination of the

ionic composition of the fluids within them and, more recently, by the use of radioisotopes

to measure the direction and rate of movement of individual ions The studies of Ramsay in

the 1950s (see reviews for references) revealed that the fluid in the tubules is isosmotic with

the hemolymph (Table 18.2) but has a very different ionic composition Particularly obvious

is the difference in potassium ion concentration, which is several times higher in the tubule

fluid than in the hemolymph The sodium ion concentration is usually lower in the fluid

than in the hemolymph, as is the case with most other ions (except phosphate) The tubule

fluid, which is produced continuously, also contains a number of low-molecular-weight

organic molecules, for example, amino acids and sugars; thus, it is broadly comparable

with the glomerular filtrate of the vertebrate kidney, though it is not produced by hydrostatic

pressure The high potassium concentration in the tubule fluid and the demonstration that the

rate at which tubule fluid is formed depends on the hemolymph potassium concentration

led Ramsay to suggest that the active transport of potassium ions is fundamental to the

production and flow of the fluid Bloodsucking insects that have just fed are exceptional

in that both potassium and sodium ions are actively transported into the tubule lumen

This modification to the basic plan is necessitated by the heavy sodium chloride load in

the plasma fraction of the vertebrate host’s blood and by the need to remove as rapidly as

possible excess water taken into the body as a result of feeding Active cation transport

is accompanied by the movement of anions (principally chloride) to maintain electrical

neutrality and by the flow of water into the tubule lumen by osmosis (Pannabecker, 1995)

Most other ions and organic molecules appear to enter the tubule fluid passively

How-ever, active transport of sulfate and of some dyes and toxic compounds (e.g., alkaloids) has

been demonstrated in some insects, notably those species that encounter these molecules

in their natural diet

It is clear that, as the tubule fluid and hemolymph are isosmotic, the tubules are not

directly concerned with regulation of hemolymph osmotic pressure Ramsay, using isolated