Mechanism of ammonia excretion in the freshwater leech Nephelopsis obscura: characterization of a primitive Rh protein and effects of high environmental ammonia

Remarkably little is known about nitrogenous excretion in freshwater invertebrates. In the current study, the nitrogen excretion mechanism in the carnivorous ribbon leech, Nephelopsis obscura, was investigated. Excretion experiments showed that the ribbon leech is ammonotelic, excreting 166.0 ± 8.6 nmol·grams fresh weight (gFW)−1·h−1 ammonia and 14.7 ± 1.9 nmol·gFW−1·h−1 urea. Exposure to high and low pH hampered and enhanced, respectively, ammonia excretion rates, indicating an acid-linked ammonia trapping mechanism across the skin epithelia. Accordingly, compared with body tissues, the skin exhibited elevated mRNA expression levels of a newly identified Rhesus protein and at least in tendency the Na+/K+-ATPase. Pharmacological experiments and enzyme assays suggested an ammonia excretion mechanism that involves the V-ATPase, Na+/K+-ATPase, and carbonic anhydrase, but not necessarily a functional microtubule system. Most importantly, functional expression studies of the identified Rh protein cloned from leech skin tissue revealed an ammonia transport capability of this protein when expressed in yeast. The leech Rh-ammonia transporter (NoRhp) is a member of the primitive Rh protein family, which is a sister group to the common ancestor of vertebrate ammonia-transporting Rh proteins. Exposure to high environmental ammonia (HEA) caused a new adjustment of body ammonia, accompanied with a decrease in NoRhp and Na+/K+-ATPase mRNA levels, but unaltered ammonia excretion rates. To our knowledge, this is only the second comprehensive study regarding the ammonia excretion mechanisms in a freshwater invertebrate, but our results show that basic processes of ammonia excretion appear to also be comparable to those found in freshwater fish, suggesting an early evolution of ionoregulatory mechanisms in freshwater organisms.

Changes in energy allocation by the predator Nephelopsis obscura exposed to differences in prey availability

Acquisition of energy and its allocation to components of bioenergetic balance were measured throughout the life cycle in three groups of Nephelopsis obscura fed one (low frequency), two (medium frequency), or three (high frequency) ad libitum meals each week at 15 and 20 °C. As feeding frequency increased, the total amounts of energy ingested, faeces plus mucus produced, somatic and reproductive growth, energy storage (total lipids), and respiration all increased. The proportion of ingested energy allocated to somatic and reproductive growth was highest for the medium-frequency and lowest for the low-frequency feeding treatment. Temperature had no effect on ingestion or faeces plus mucus production, but the respiration rate was higher and the growth rate lower at 20 than at 15 °C. The proportions of ingested energy allocated to growth and storage were the same in the high- and medium-frequency feeding treatments at both temperatures. In the low-frequency feeding treatment, the proportion of energy allocated to storage was higher at 15 °C, but proportionally more energy was allocated to somatic growth and less to reproductive growth or lipid storage at 20 °C. In the high- and medium-frequency feeding treatments, the relative amount of energy allocated to storage was constant, i.e., the ratio of reserves to structural tissues was the same, at both temperatures.

Changes in reproductive potential of the leech Nephelopsis obscura (Erpobdellidae) as biomarkers for cadmium stress

The effects of exposure for 4–24 days to 0, 58, 145, 290, 435, and 580 μg∙L−1 cadmium on the reproductive potential of the freshwater predatory leech Nephelopsis obscura were investigated. The numbers of ova and spermatozoa per unit biomass were significantly reduced with increasing cadmium concentration and exposure time, as were the masses of the ovisacs, testisacs, and epididymis plus cornu. It is concluded that these variables are effective biomarkers for identifying sublethal effects of direct importance to population dynamics.