Arthur W. Martin

Iron binding proteins and their roles in the tobacco hornworm, Manduca sexta (L.).

SummaryManduca sexta larvae accumulate large amounts of iron during their larval feeding period. When59Fe was fed to 5th instar larvae, it was evenly distributed among the hemolymph, gut and carcass until the cessation of feeding. By pupation 95% of the labelled iron was found in the fat body. In the adult a significant portion of this iron was found in flight muscle.Studies of the hemolymph disclosed two ironcontaining proteins. The first was composed of a single polypeptide chain of 80 kD, containing one atom of iron. This protein bound ionic iron in vitro and was able to transfer this iron to ferritin when incubated with fat body in vitro. Therefore, it appeared to serve a transport function. The second protein had a molecular weight of 490 kD with subunits of 24 and 26 kD and contained 220 μg of iron/mg protein. Its chemical and ultrastructural characteristics were those of ferritin. These studies demonstrate the presence of both a transport protein and a unique circulating ferritin inManduca sexta, the latter serving a storage function during development and possibly also a transport function.

Circulation in the cephalopod, Octopus dofleini.

Abstract 1. 1. Simultaneous recordings of pressure from the cephalic aorta, the afferent and efferent branchial vessels and the vena cava cephalica have been made in unanethetized, unreastrained cephalopods, Octopus dofleini . 2. 2. The frequency of the systemic heart varied between 8–18 beats/min at a water temperature of 7–9°C. The aortic systolic pressure varied during normal conditions between 45–70 cm of water with a pulse pressure of 20 cm of water. The pressure level in the afferent branchial vessels ranged from 25 to 50 cm of water systolic and about 15 cm of water diastolic pressure. Similar values in the efferent branchial vessels were 10–25 cm of water systolic and 5–15 cm of water diastolic. In the vena cava cephalic the pressure ranged under normal conditions between 0–17 cm of water with a pulse pressure of 3–5 cm of water. 3. 3. The pressure recordings indicate that the ctenidia contract actively in a rhytmic fashion, promoting the propulsion of blood. The pressure changes in the vena cava cephalica are thought to be passively mediated from pressure changes created by the respiratory movements. 4. 4. During exercise there is a marked increased in both pulse pressure and diastolic pressure in the aorta. The heart sometimes showed great acceleration during exercise. 5. 5. Experiments with infusion of sea water into the vascular system demonstrated a capacity for accomodation of large volumes without noteceable disturbance of the general circulation. 6. 6. Pressure recordings in a symmetrical arrangement in the branchial vessel indicate a crucial importance of the nervous system for coordination of activity in the octopus vascular system. 7. 7. The haemodynamics of the cardiovascular system in Octopus dofleini are discussed.

Male Reproductive Tract, Spermatophores and Spermatophoric Reaction in the Giant Octopus of the North Pacific, Octopus dofleini martini

The male reproductive tract of Octopus dofleini martini lies enclosed in the genital bag, inside the mantle cavity. It consists of the testis, vas deferens proximale, spermatophoric gland system I (seminal vesicle), spermatophoric gland system II (prostate), vas deferens distale, spermatophoric sac (Needham’s sac) and the terminal spermatophoric duct. The spermatozoa, which upon leaving the testis are as yet not encased, pass first into the vas deferens proximale, which in its gross appearance resembles the epididymis; they then traverse the two tubular spermatophoric gland systems I and II, where they become encased into the spermatophores. Subsequently the spermatophores pass through the vas deferens distale into the spindle-shaped spermatophoric sac, and from there they enter singly the terminal spermatophoric duct consisting of the diverticulum and terminal organ or ‘penis’. The slender cylindrical body of the spermatophore is about 1 m long and consists of two parts, approximately equal in length. The thicker ‘proximal’ ‘male-oriented’ portion, which emerges first from the orifice of the ‘penis’, contains the tightly coiled sperm rope suspended in a viscous and transparent fluid, the spermatophoric plasma; the thinner ‘distal’ ‘femaleoriented’ half of the spermatophore is taken up by the rod-shaped, hyaline core of the ejaculatory apparatus. In between is located the cement body, and the amber-coloured cement liquid. At the distal end of the spermatophore the outer coating forms a cap and a filamentous appendage, the cap-thread. Chemical analyses were performed on spermatozoa, spermatophoric plasma, cement liquid, outer tunic and the hyaline core of the ejaculatory apparatus, obtained from freshly recovered spermatophores. Glycogen was identified as a major constituent of spermatozoa. The extraordinarily high dry-weight content of spermatophoric plasma (nearly 30%) was shown to be largely due to bound amino sugar, carbohydrate, peptide and protein. A peptide separated from the spermatophoric plasma by ultrafiltration was found to be made up to a great extent of aspartic acid and serine. The outer tunic, a tough and elastic membrane, which envelops the body of the spermatophore, was shown to consist mostly of a protein which is rich in proline, lysine, aspartic acid and threonine. The mechanics of the spermatophoric reaction in vitro have been studied in spermatophores extracted manually from the male octopus and placed in sea-water. The complete spermatophoric reaction under such conditions lasted 1 to 2 h. During that interval the sperm rope gradually advanced a distance of about 1 m, from the proximal towards the distal end of the spermatophore. The terminal phase of this process involved an evagination of the ejaculatory apparatus, followed by a rapid movement of the sperm rope; as the sperm rope entered the end portion of the spermatophore, the latter ballooned out into an egg-shaped bladder. Among the factors which contribute to the formation of the spermatophoric bladder, the most important ones are (i) the elasticity of the membranes of the spermatophore, (ii) the extrusion, and subsequently evagination, of the ejaculatory apparatus, and (iii) the influx of sea-water into the spermatophore’s body which causes an approximately fivefold increase in the volume of spermatophoric plasma. Concomitantly with the uptake of sea-water, the dry weight of the spermatophorie plasma declines but the sodium chloride concentration increases. However, the osmolality of the spermatophoric plasma, as assessed by freezingpomt depression, is not altered during the spermatophoric reaction. Events at copulation, that is under conditions in vivo, closely resembled those observed in spermatophores undergoing a spermatophoric reaction in vitro. An interval of 2 to 3 h usually elapsed from the time when the male, using his hectocotylized arm, mserted mto the female the distal (female-oriented) end of a spermatophore, to the moment of the males withdrawal. After accomplished copulation two spermatophores were usually found firmly lodged in the two oviducts. The sperm-free remnants of the spermatophore bodies dangled free from the orifices of the oviducts. Upon dissection of recently mated females the spermatophoric bladder was usually found within the oviduct, held firmly in position by the evaginated ejaculatory apparatus.

Notes on animal weight, cameral fluids, swimming speed, and color polymorphism of the cephalopod Nautilus pompilius in the Fiji Islands

Forty-six specimens of Nautilus pompilius Linnaeus were captured in depths varying between 100 and 500 m outside of the fringing reef near Suva, Fiji Islands. Thirty- eight of the specimens were male. Air weight per individual varied between 347 and 630 g. Sexual dimorphism in size is indicated, since mature shell modifications (approximated septa, blackened aperture) were present in two females weighing about 350 g (soft parts plus shell) and one weighing slightly over 400 g; the smallest male showing mature shell mod- ifications weighed 496 g. All newly captured specimens were heavier than seawater, with mean weight in seawater of 1.87 g determined for twenty-five specimens. Total volumes of cameral liquid ranged between 13.5 and 0 ml. Thirteen of twenty-five sampled specimens showed less than 1.0 ml of cameral liquid from all chambers. Average cameral liquid os- molarity was lower than that observed in sampled populations of N. macromphalus from New Caledonia and N. pompilius from the Philippine Islands. Maximum swimming rates were 0.25 m/sec. N. pompilius exhibits two common color polymorphs.

Blood pressure in the tarantula,Dugesiella hentzi

Summary1.Heart rates in intact spiders at rest were 35/min and after 3 min exercise 77/min. Recovery to the initial rate required 1/2 hr. In cannulated animals heart rates ranged from 44/min at rest to 116/min after a struggle. Heart rate was relatively independent of internal pressure.2.Variations in heart rate and amplitude occurred spontaneously and in response to various stimuli (Fig. 6).3.Systole averages 68% of total beat duration through a range of heart rates from 21–116 min (Fig. 4).4.Intraventricular pressures 10 min after cannulating averaged 22/13 mm Hg, but fell to 12/8 mm Hg after 10 min in the dark (Fig. 5). A maximum pressure of 102 mm Hg was measured in one animal. The pressure recorded in the heart is generated in part by the heart and in part reflects abdominal tension.5.Resting pressures in any 2 legs were always identical and were above that in the prosoma.6.Resting pressures in the prosoma and lateral sacs are intermediate to heart systolic and diastolic pressures which in a relaxed animal were 12/8 mm Hg (Fig. 11).7.At rest a gradient of only a few mm Hg propels the blood through the lung books from the lateral sacs to the pulmonary veins; a gradient of 1–2 mm Hg exists between the pulmonary vein and the heart in diastole.8.General abdominal pressure is above pulmonary vein pressure suggesting a need for a mechanism to maintain patency of these vessels.9.During a struggle a maximum pressure of 480 mm Hg was measured in the prosoma.10.High pressures were recorded in the lateral sacs which communicate freely with the prosoma but were not transmitted to the heart.11.Pressures of 40–60 mm Hg were measured in the prosoma and legs during wialking (Fig. 18) and delivery of blood through the pedal arteries probably continues at this level.12.Withdrawal of blood causes an immediate decrease in heart pressure. Within min some recovery occurs presumably because of muscular adjustments (Fig. 19).

Blood and fluid balance of the common tarantula, Dugesiella hentzi

Summary1.Blood of Dugesiella hentzi, the common tarantula of the United States, was analyzed. Protein concentration of serum averaged 74 mg/ml with hemocyanin the major constituent since the Cu/protein ratio of 0.00175 is close to that of purified hemocyanin from other arthropods. Concentrations of non-protein nitrogen, glucose, and total anthrone reactive material were 0.32, 0.04, and 0.13 mg/ml respectively. Freezing point depression of the serum was 0.75° C and pH ranged from 7.25 to 7.35.2.Blood volumes of males and females expressed as percent of body weight averaged 19.65 and 18.10% respectively. Water content of females was about 73.2% of body weight. Fat content averaged 10.3% of body weight. Exoskeleton represented 5.8% of body weight with a water content of 40%. A value of 74.7% was calculated for intracellular water.3.Evaporation rates were determined. During the first hour of exposure to moving dry air, 0.168 mg/cm2/hr was lost at 20° C increasing to 0.915 mg/cm2/hr at 40° C. By the fifth hour of exposure these losses had decreased to 0.088 mg/cm2/hr at 20° C and 0.674 mg/cm2/hr at 40° C (Fig. 1).4.Drinking habits in the laboratory were observed. Fed regularly, 2 crickets per week, the spiders usually drank once weekly, soon after eating. Volumes taken varied from a few mg to more than 1 g. Fasted spiders drank somewhat more, tending to maintain constant weight; the contribution of metabolic water was only 5% of the total.5.Desiccation for periods to 1 week resulted in approximately 20% decreases in both blood volume and body weight. This represents a loss of 27% of the total body water and a decrease of only 9.4% in intracellular water.6.After removal of 15–50% of the total blood volume, entry of fluid diluted the remaining blood and the normal volume was restored by about 3 weeks after bleeding. Synthesis of blood protein was slow. Not until after two months was the protein level back to the initial level. Protein synthesis averaged 0.0064 mg/g/day when 20–36% of the initial blood volume had been removed.7.Results suggest that when a loss of body contents occurs from bleeding, egg laying or withholding of food, these animals replace the lost volume with water.

Immunological and biological characteristics of the vasotocin-like activity in the head ganglia of gastropod molluscs

Extracts of cerebral and pleuro-pedal ganglia from two terrestrial slugs, Ariolimax columbianus and Limax maximus, and from the marine opisthobranch, Aplysia californica, contain immunoreactivity resembling that of a vasotocin or vasopressin. Radioimmunoassays using several antisera indicate that the immunoreactivity is not due to vasotocin, vasopressin, or any other known naturally occurring neurohypophyseal peptide. Immunoreactivity of extracts on a relatively nonspecific vasopressin antiserum is well correlated with activity on antidiuretic assays on rats. Both immunoreactivity and antidiuretic activity are adsorbed onto bovine neurophysin affinity columns. Thus these extracts contain one or more peptides that closely resemble the vertebrate antidiuretic hormones, vasotocin and vasopressin, both immunologically and pharmacologically. The amounts of immunoreactivity and antidiuretic activity in ganglion extracts do not appear to change during dehydration and rehydration. Although both ganglionic extracts and vasotocin stimulate exudation of fluid across the slug body wall, the present experiments provide no evidence that the vasotocin-like material(s) in these ganglia may participate as neurotransmitters or hormones in the regulation of fluid balance. This remains an attractive hypothesis.

Characterization of an invertebrate transferrin from the crabCancer magister (Arthropoda)

SummaryAn iron binding protein was isolated from crab blood and tested for its chemical and functional behavior.1.The protein has a molecular weight of 150,000±10,000 and consists of a single polypeptide chain. The iron content was found to be two molecules iron per molecule protein; the presence of a 52-fold molar excess of copper did not alter the iron binding properties.2.Spectroscopical analysis carried out in the presence of bicarbonate yielded two absorption maxima at λ=276 and λ=465 nm.3.The59Fe-tagged crab protein did not yield its iron to human apotransferrin when incubated in human plasma. When incubated with rat reticulocytes, the crab iron protein delivered its iron for hemoglobin and ferritin synthesis. This delivery can be completely abolished by adding an excess of rat transferrin.4.When injected into rats in vivo, the crab protein delivered its iron to erythroid and non-erythroid tissue. The amount delivered to the erythroid marrow was shown to depend on iron requirements of that tissue.5.When injected as59Fe-tagged protein into a crab, half-time disappearance of the radioiron is reached between 7 and 9 h. In the liver and in the carapace lining, the radioiron released is found in other iron binders.6.Except for the significantly larger molecular weight, the crab iron binding protein fulfills all the criteria for transferrin. The findings negate the concept that transferrin is a newcomer in the evolutionary scene and to be found only in the phylum Chordata. Thus the need for a specific iron transport protein of the transferrin class is independent of the achievement of hemoglobin as a means to accomplish oxygen transport.

Hypertrophy as a response to denervation in skeletal muscle

Summary1.The effect of denervating the hemidiaphragm of the quokka (Setonyx brachyurus), hamster, guinea pig and bat (Myotis pallidus) was determined. The hemidiaphragm of the quokka neither hypertrophied nor atrophied during the period of 171 days (Fig. 1). The hemidiaphragm of the hamster exhibited a slight hypertrophy, about 5%, during the first week and by 28 days atrophy of 13% had occurred (Fig. 2). The hemidiaphragm of the guinea pig exhibited no hypertrophy but by 30–35 days atrophy of 15% had occurred (Fig. 3). In the bat levels of hypertrophy to 30% occurred but the average during the first 2 weeks was only 8%. Atrophy of 9.5% had occurred by 3 weeks (Fig. 4A).2.Bats kept at 2–3° C or 10° C after denervation of the hemidiaphragm exhibited average extents of hypertrophy comparable to that occurring at room temperature (Fig. 4 B).3.Various limb muscles were denervated for comparison. The biceps brachii muscles of the quokka atrophied 15% by 2 weeks and 35% by 35 days (Fig. 1). The gastrocnemius-soleus muscles of the hamster had atrophied 40% by 14 days, 55% by 28 days, while the plantaris muscle atrophied more slowly, 31% and 46% by 14 and 28 days respectively (Fig. 2). The major wing extensors of the forearm, the extensor carpi radialis longus and extensor carpi radialis brevis, exhibited a significant hypertrophy in the bat averaging 4.3% during the first 24 days (Fig. 5). No atrophy occurred during a period up to 42 days. These muscles are maintained in the extended position by folding of the wing after denervation.4.The report of Gutmann (1960) that hypertrophy of the anterior tibial muscle of the rat occurred during the first 24 hours after denervation as evidenced by an increase in non collagenous protein nitrogen was not confirmed. No increase in this component or in wet or dry weight was observed (Table 2).5.The report of Feng et al. (1962) that hypertrophy of the anterior latissimus dorsi and atrophy of the posterior latissimus dorsi of the chicken occurs after denervation was confirmed. The teres minor of the chicken, which is stretched by drooping of the wing after denervation, exhibited hypertrophies to 62% during the first 30 days. Atrophy was not observed (Fig. 6).6.The anterior latissimus dorsi of the pigeon exhibited increases in dry weight to 43% during the first month after denervation. The deltoideus major, deltoideus minor, and scapulotriceps of the pigeon exhibited atrophies of 45% by 20–23 days (Fig. 7). When the wings of the pigeon were supported by a cast atrophy of the denervated anterior latissimus dorsi occurred, reaching 38% by 18–20 days (Fig. 8).

D(—)-Lactic Acid and D(—)-Lactate Dehydrogenase in Octopus Spermatozoa

The spermatozoa of Octopus dofleini martini produce anaerobically D(—)-lactic acid and possess a very active D(—)-lactate dehydrogenase. In this respect, while resembling certain microorganisms, they differ strikingly from mammalian spermatozoa which produce L(+)-lactic acid and contain L(+)-lactate dehydrogenase.