EXCRETION • Eliminates interaction of harmful substances with cells & tissues (e.g., nitrogenous substances, allelochemicals) & maintains internal environment.

Wastes often assoc’d with water; therefore excretion closely assoc’d with osmoregulation & water balance. Insects usually need to conserve water - must excrete but conserve water. Excretory system helps maintain homeostasis - maintenance of constancy of internal environment of cells and tissues. Metabolism of lipids & carbohydrates produce CO2 + water - not toxic & easily rid. But metabolism of proteins & nucleic acids ⇒ nitrogenous waste products that must be excreted. Excretion involves Malphigian tubules (MT) & hindgut working together; MT collect filtrate from hemolymph and pass this primary urine to hindgut; additional components excreted by hindgut & some reabsorbed into hemolymph.

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Ammonia Excretion • In biological systems XS nitrogen readily forms NH3. Free NH3 can’t be stored in tissues or cells (strong base & toxic) - must be rapidly excreted or transformed into less toxic molecule. • If water available, NH3 can be excreted as free base or NH4+ salt; animals that secrete thus - ammoneotelic. NH3 major excretory product for some larval Diptera that live in wet conditions, e.g, Calliphora blowfly larvae, or some aquatics - NH3 rapidly diluted in environment. Also some terrestrial insects, e.g., Periplaneta - excrete NH3 as major product. But needs a lot of water. • Some NH3 can be recycled into AA synthesis by formation of glutamate from αketoglutarate and then glutamine. • Most animals synthesize NH3 into less toxic form - urea (mammals) or uric acid (birds, reptiles, insects). These less toxic than NH3 & use less water for excretion. Uric Acid Synthesis and Secretion • Uric acid rel insoluble in water and therefore doesn’t reach toxic levels. Also, can precipitate from solution in MT & hindgut & not contribute to osmotic pressures. • But biosynthesis of uric acid costly. In birds (& prob insects), synth 1 mole uric acid from NH3 requires 8 moles ATP. But rids body of 4 Ns. Fat body primary site for synth of uric acid. Final steps synthesis involves conversion of hypoxyanthine and xanthine to uric acid (catalyzed by xanthine dehydrogenase, not xanthine oxidase as in vertebrates). Dehydrogenase requires proton acceptor such as NAD+ or FAD+. This can generate NADH or FADH, which thru ETS recovers, respectively, 6 or 4 moles ATP/mole uric acid. • Many insects excrete nitrogen as allantoin, a breakdown product of uric acid catalyzed by enzyme uricase; also allantoic acid excreted in some insects. • Animals that excrete most or all of nitrogen as uric acid called uricotellic.

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Malphigian Tubules • Long, tubular structures usually arising (proximal end) at junction of mid- and hindgut; terminate (distal end) blindly in hemocoel. From 2 - >100 tubules in various spp; Collembola, Aphidae, and some Thysanura lack MT – other cells/glands take over function. • Walls consist of single cell layer of epithelial cells, usually large with polyploid nuclei, that are differentiated along length of tubule. On hemolymph side, cells covered by basement membrane (with extensive infoldings) with numerous trachea supplying oxygen (high metabolic demand). Membrane on lumen side have brush border of microvilli rich in endoplasmic reticulum & mitochondria. Intermediate region of cell has numerous Golgi bodies, RER and vacuoles.

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Nation, 2002 Tubules surrounded by longitudinal muscles which promote coiling movements that assist flow of fluid and increase contact with hemolymph. In some Leps & Coleops, distal ends of MT embedded in wall of rectum (known as crytptosolenic or cryptonephridial tubules) – prob. aids water conservation. MT initiate excretion process. Fluid, ions, & waste (primary urine) are transported into tubule lumen, and travel from distal to proximal end of tubules, where enters alimentary canal and mixes with end products of digestion (from midgut). Primary urine passes through hindgut and is modified by resorption to produce secondary urine (expelled). Tubules equivalent of mammalian kidney. However, excretion by MT achieved exclusively by secretion of ions & organic molecules from hemolymph into tubule lumen; c.f. kidneys, most of contents of kidney tubule derive from plasma filtered into lumen (at Bowman's capsule). As in mammalian kidney, contents of lumen modified as pass thru the MT and hindgut.

Formation of Primary Urine • Primary urine - filtrate of hemolymph; contains most of small ions & molecules (sugars, AAs) that in hemolymph. The urine:hemolymph conc ratio for many of the filtered substances is ca. 1: indicates passive movement across tubule membranes. Some components actively secreted; their ratio is >1.

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Initial expts showed primary urine iso-osmotic with hemolymph but [K+] 20X greater ⇒ water follows K+ transport. Urine formation in MT relies on vacuolar proton pump in apical membranes of MT cells - actively secretes H+ into tubule lumen against electrochemical gradient. Pump consists of V1 protein complex in cytoplasm of principal cells in MT & an ion channel formed by V0 transmembrane complex embedded in lipid bilayer of apical membrane. Pump causes tubule lumen to become +ve (up to 30mV) to the hemolymph and creates pH gradients across apical membrane of principal cells. H+ gradient provides energy for antiporter mech. (exchanges K+ for H+); net result - K+ secreted into MT lumen & conc’d against electrochemical gradient. In some insects that take blood meal rich in Na+ (e.g., mosquitoes), Na+ actively transported by a pump. Formation of urine volume highly dependent on [K+] in hemolymph. Cations such as K+ and /or Na+ & probably anions must enter MT cells from hemolymph for secretion to continue. Secretion of cations across apical membrane is electrically coupled with Cl- transport (thru ion channels). Water follows ions (osmosis) Water & some solutes in hemolymph may diffuse by paracellular (between adjacent cells) pathway; some toxins & metabolites (incl uric acid) actively transported across cells by transcellular route. Sugars, AAs & allelochemicals in hemolymph follow osmotic gradient created by transport of K+ and other ions.

Klowden, 2002 If unmodified primary urine excreted – insects depleted of K+ and water. But, hindgut, selectively reabsorbs some substances back into hemolymph, leaves others in lumen, & actively secretes some substances into hindgut lumen.

Secretion and Reabsorption in Ileum. • Ileum - most anterior part of hindgut. In locusts, ileum is major site for isosmotic fluid reabsorption; active Na+ and Cl- reabsorption and active secretion of proline. Driving mech. for ion and water reabsorption in ileum is electrogenic (produces electrical impulses) Cl- pump. • Ileum plays major role in acid-base balance by secretion of H+ into lumen,

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formation of NH4+ and reabsorption of HCO3-. Metabolism of reabsorbed nonessential AAs present in urine releases energy for ATP synth for active reabsorption; ca. 80% of NH3 produced in epithelial cells from metab of these AAs is transported into lumen where excreted as NH4+.

Reabsorption in Rectum • Rectum final & major site for reabsorption of ions, water & nutrients; reabsorbs against strong osmotic gradients; produces conc hyperosmotic excreta in insects. Works opposite to MT; transports water & ions in gut lumen to hemolymph. • Rectal cuticular lining has greater permeability than foregut cuticular lining. In rectum, small groups of enlarged epithelial cells (rectal cells, rectal pad cells, or rectal papillae cells) have modifications for reabsorption. Usually absent in fluidfeeding insects that do not reabsorb fluid from primary urine. • Electrogenic Cl- pump on lumen side of cell membrane drives process, by moving Cl- from gut lumen to hemolymph; pump under influence of neuropeptide chloride transport stimulating hormone (CTSH) (from Corpora cardiaca). A Na+/K+-ATPase mediates transport of Na+ into hemolymph. K+ reabsorption passive; ion channels open thru electrical coupling. • Water & dissolved solutes from rectal lumen follows osmotic gradient created by ion absorption. As water reabsorbed, excreta in rectum becomes pasty or dry.

Klowden, 2002 Rectal epithelial cells actively reabsorb AAs from lumen & metabolize them to produce ATP for the pumps.

Hormonal Control of Excretion and Osmoregulation • Although insect excretory system conserves water, also able to rid itself of excess water when needed. Insects increase their urine production using diuretic (peptide) hormones. Insect diuretic hormones fall into two classes: 1. Those similar to vertebrate corticotropin-releasing factor (CFC-peptides); 30-46 AAs; stimulate adenylate cyclase and raise cAMP levels. Causes opening of Na+ channels in MT. 2. Smaller kinins (6-15 AAs); activate Ca2+ signaling pathway. Named according to genus isolated from; e.g., achetakinins from Acheta domesticus. Cause K+ transport to increase and Na+ to decrease. • MT secretion typically controlled by interaction of several compnds - effects of

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diff compnds can be synergistic, additive or even inhibitory (because diff mechs). Cryptonephridial Systems • Many families of Coleopts, Leps and some sawfly larvae have arrangement in MTs in which distal ends of tubules enveloped within membrane & held close to rectum surface - cryptonephridic tubule system; appears to help efficient conservation of water; used by insects living in v dry environments.

Klowden, 2002 Cryptonephridic tubules don’t penetrate lumen of rectum; lie on outer surface encased within perinephric chamber bounded by perinephric membrane. Perinephric membrane composed of thin, elongate cells that seal tubules from hemocoel and hemolymph at point of contact with gut. Tubule does not terminate after contacting rectum but typically makes many loops & convolutions running radially around rectum and looping back and forth. In most Coleopts, a crytonephridic tubule in outermost layer makes contact with outer perinephric membrane thru single highly modified cell of tubule called leptophragma cell ( Leps don’t have these). In both Coleops & Leps, extent of layering of crytponephridial loops on the rectum correlated with dryness of food - more loops, etc, drier food (better water conservation?). Physiological functioning of the cryptonephric complex not well-elucidated.

Storage Secretion - Sequestration of wastes inside body • Because uric acid insoluble can be stored without adverse effects on physiology; some cockroaches store uric acid hemolymph or in specialized urate cells in fat body – can utilize store in times of stress. Also male Blatella germanica store it in accessory glands - material transferred to female during copulation. • In some Leps, fat body changes from excreting uric acid to storing it. Occurs before pupation. Stores transported to rectum shortly before eclosion (and eliminated as meconium).

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Reading: Beyenbach KW. (2003). Transport mechanisms of diuresis in Malpighian tubules of insects. J Exp Biol. 2003 Nov;206(Pt 21):3845-56.

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