Mark J. Snyder

Cytochrome P450 enzymes in aquatic invertebrates: recent advances and future directions

A variety of enzymes and other proteins are produced by organisms in response to xenobiotic exposures. Cytochrome P450s (CYP) are one of the major phase I-type classes of detoxification enzymes found in terrestrial and aquatic organisms ranging from bacteria to vertebrates. These enzymes metabolize a wide variety of substrates including endogenous molecules (e.g. fatty acids, eicosenoids, steroids) and xenobiotics (e.g. hydrocarbons, pesticides, drugs). Aquatic invertebrates, especially those in marine habitats, occupy every aspect of the environment, from above the surface (intertidal) to below the sediments. In turn, they have extremely diverse physiologies and are exposed to a vast array of potential toxicants. Aspects of aquatic invertebrate cytochrome P450 enzymes have been studied for the last 25 years. In a few phyla, P450 activities have been measured and are responsive to xenobiotic exposures. Until the last several years, little progress had occurred in the identification of P450 gene diversity in aquatic invertebrates. Molecular biology tools have greatly aided this search, and are likely to identify as much diversity for this protein superfamily as is present in higher marine and terrestrial organisms. Recent work has expanded our knowledge of the CYP superfamily, and new developments will rapidly advance the usefulness of these genes into such fields as biomarker research. Advances of the last decade are reviewed and insights are presented from related insect studies.

Osmotic Induction of Stress-Responsive Gene Expression in the Lobster Homarus americanus

The American lobster, Homarus americanus, encounters osmotic stress throughout its life cycle. To understand the molecular basis of osmotic stress responses in vivo, we used homologous cDNA probes to characterize the mRNA patterns of lobster HSP70 (=70-kDa heat-shock protein), HSP90 (=90-kDa heat-shock protein), and polyubiquitin during hypo- and hyper-osmotic stress in abdominal muscle and hepatopancreas (a digestive tissue) at 30, 60, and 120 min of osmotic stress. Hypo- and hyper-osmotic stress significantly increased the levels of the mRNAs encoding HSP70 and HSP90 in abdominal muscle. Hyper-osmotic stress increased HSP90 mRNA levels in hepatopancreas, but hypo-osmotic stress did not. Both abdominal muscle and hepatopancreas exhibited significant changes in polyubiquitin gene expression during osmotic stress. In abdominal muscle, polyubiquitin mRNA levels increased during both hypo- and hyper-osmotic stress. Hepatopancreas, however, showed a significant elevation in polyubiquitin mRNA only during hypo-osmotic stress.

Environmental endocrine disruption in decapod crustacean larvae: hormone titers, cytochrome P450, and stress protein responses to heptachlor exposure.

A variety of enzymes and other proteins are produced by organisms in response to xenobiotic exposures. Cytochrome P450s (CYP) are one of the major phase I-type classes of detoxification enzymes found in terrestrial and aquatic organisms ranging from bacteria to vertebrates. One of the primary functions of stress proteins (HSPs) is to aid in the recovery of damaged proteins by chaperoning their refolding. These and other biomarkers of xenobiotic exposure and resulting effects have not been studied in crustacean larvae. This information is of potential importance for environmental management and risk assessment. In this work, we have given Homarus americanus larvae single 24 h exposures to the cyclodiene pesticide heptachlor, a known environmental endocrine disruptor (EDC) on different days of the 1st larval instar. We followed these larvae during the first larval stage for effects on timing of ecdysis to 2nd stage, ecdysteroid molting hormone titers, and alterations in the levels of cytochrome P450 CYP45 and HSP70 proteins. Delays in ecdysis were correlated with alterations in ecdysteroid levels. This result provides clues that this pesticide may function as an environmental endocrine disruptor in crustaceans. CYP45 and HSP70 levels were significantly elevated for several days following heptachlor exposure. The elevation in HSP70 was prolonged depending on the day of pesticide exposure and this was directly related to the increase in mortality. These results demonstrate the utility of these measurements as potential biomarkers in crustacean larval developmental toxicology and EDC effects research.

Competition for space among sessile marine invertebrates: changes in HSP70 expression in two Pacific cnidarians.

The role of stress proteins—either constitutive (HSC) or inducible (HSP)—of the HSP70 family in intra- and interspecific competition for space was examined in two sessile Pacific cnidarians. Anthopleura elegantissima, an intertidal anemone, and Corynactis californica, a subtidal corallimorpharian, express HSP70 in the absence of apparent physical stress. HSP70 protein expression is concentrated in the tentacles of A. elegantissima when the animal is exposed to contact with other benthic organisms. Under the same conditions, however, HSP concentrations are similar in the body and tentacles of C. californica. When two different clones of A. elegantissima interact in the field, the outside polyps (warriors) express more HSP70 than the inside ones (2.4 versus 0.6 ng HSP70/μg Protein). When different C. californica clones interact, HSP70 expression in the outside and inside polyps is similar (1.5 versus 1.8 ng HSP70/μg P) and is fairly constant in the corallimorpharian in the different interspecific encounters. HSP70 expression is related to the different kinds of aggression encountered by both cnidarians. HSP70 expression may be involved in the recovery of tissues damaged by the allelochemical, cytotoxical, or corrosive substances produced by different enemies. C. californica clones appear prepared for war, as evidenced by the high constant expression of HSP70 in the polyps. A. elegantissima exhibits differential HSP70 expression depending on the identity of each neighboring intra- or interspecific sessile competitor. We propose that stress proteins can be used to quantify space competition or aggression among sessile marine invertebrates.

Thermal acclimation and stress in the American lobster, Homarus americanus: equivalent temperature shifts elicit unique gene expression patterns for molecular chaperones and polyubiquitin

Abstract Using homologous molecular probes, we examined the influence of equivalent temperature shifts on the in vivo expression of genes coding for a constitutive heat shock protein (Hsc70), heat shock proteins (Hsps) (Hsp70 and Hsp90), and polyubiquitin, after acclimation in the American lobster, Homarus americanus. We acclimated sibling, intermolt, juvenile male lobsters to thermal regimes experienced during overwintering conditions (0.4 ± 0.3°C), and to ambient Pacific Ocean temperatures (13.6 ± 1.2°C), for 4–5 weeks. Both groups were subjected to an acute thermal stress of 13.0°C, a temperature shift previously found to elicit a robust heat shock response in ambient-acclimated lobsters. Animals were examined after several durations of acute heat shock (0.25–2 hours) and after several recovery periods (2–48 hours) at the previous acclimation temperature, following a 2-hour heat shock. Significant inductions in Hsp70, Hsp90, and polyubiquitin messenger RNA (mRNA) levels were found for the ambient-acclimated group. Alternatively, for the cold-acclimated group, an acute thermal stress over an equivalent interval resulted in no induction in mRNA levels for any of the genes examined. For the ambient-acclimated group, measurements of polyubiquitin mRNA levels showed that hepatopancreas, a digestive tissue, incurred greater irreversible protein damage relative to the abdominal muscle, a tissue possessing superior stability over the thermal intervals tested.

Effects of eyestalk ablation on larval molting rates and morphological development of the american lobster, Homarus americanus

Larvae of the lobster, Homarus americanus, displayed abbreviation of subsequent molt intervals in response to bilateral eyestalk ablation at the 2nd stage. The interval in which the ablation occurred (2nd stage) was occasionally shortened. However, the 3rd stage molt interval was always abbreviated. At the 4th stage the ablated larvae were larger than intact controls. Removal of the last pair of swimming legs, as a control for the trauma of eyestalk ablation, slightly decreased growth but had no effect on molt interval lengths. Eyestalk removal sometimes resulted in 4th stage larvae with characteristics intermediate between normal 3rd and 4th stages. Eyestalkless larvae that were reared communally and fed frozen Artemia formed significant numbers of these intermediates. Eyestalkless larvae reared individually and fed live Artemia nauplii rarely formed intermediates. These results imply that although eyestalks may be involved in the regulation of both the timing and growth increment of larval molting, they...

Metabolism and Excretion of Injected [3H]-Ecdysone by Female Lobsters, Homarus americanus

The dynamics of ecdysteroid metabolism and excretion were followed in adult lobsters, Homarus americanus. Females at five different molt stages were injected with [3H]-ecdysone. Levels of [3H]-20-hydroxyecdysone (20E), converted from [3H]-ecdysone, rose rapidly and remained significantly higher in premolt stages D0 and D1. In contrast, significant increases in the levels of highly polar ecdysteroid metabolites (HP) occurred primarily in stages A and C. Changes in the hemolymph levels of 20E and HP in hemolymph over the molt cycle suggest additional metabolic mechanisms by which the titers of active molting hormones can be regulated. Excretion of [3H]-ecdysteroids was slower during early premolt stages D0 and D1, suggesting that this reduced rate may be an additional mechanism for regulating ecdysteroid titers. Study of [3H]-ecdysteroids indicated that metabolism proceeds primarily to HP that are excreted in the urine with unaltered ecdysteroids. An additional ecdysteroid metabolic route was found in the midgut gland; this route removes ecdysteroids from the hemolymph and transforms them into apolar metabolites prior to their excretion in the feces. This route is similar to that previously found for ingested [3H]-ecdysone, which was converted to apolar conjugates without further absorption.

Molt cycle–dependent molecular chaperone and polyubiquitin gene expression in lobster

Abstract Lobster claw muscle undergoes atrophy in correlation with increasing ecdysteroid (steroid molting hormone) titers during premolt. In vivo molecular chaperone (constitutive heat shock protein 70 [Hsc70], heat shock protein 70 [Hsp70], and Hsp90) and polyubiquitin messenger ribonucleic acid (mRNA) levels were examined in claw and abdominal muscles from individual premolt or intermolt lobsters. Polyubiquitin gene expression was assayed as a marker for muscle atrophy. Both Hsc70 and Hsp90 mRNA levels were significantly induced in premolt relative to intermolt lobster claw muscle, whereas Hsp70 mRNA levels were not. Hsp90 gene expression was significantly higher in premolt claw muscle when compared with abdominal muscle. Polyubiquitin mRNA levels were elevated in premolt when compared with intermolt claw muscle and significantly elevated relative to premolt abdominal muscle.

EFFECTS OF SINUS GLAND EXTRACTS ON LARVAL MOLTING AND ECDYSTEROID TITERS OF THE AMERICAN LOBSTER, HOMARUS AMERICANUS

Eyestalk ablation of 2nd stage larvae of the lobster, Homarus americanus, resulted in an accelerated rise in whole larval ecdysteroid titers during the 3rd stage. The appearance of both the premolt ecdysteroid peak and the subsequent ecdysis were accelerated by eyestalk removal during the previous molt interval. Replacement therapy, using sinus gland (SG) extracts from juvenile lobsters injected into 3rd stage larvae (ablated during the 2nd stage), delayed the next molt. This delay was significantly longer than for larvae injected with non-sinus gland (NSG) eyestalk tissue extracts. Sinus gland extracts also decreased ecdysteroid titers of ablated larvae within 12 h. Basal levels were maintained in SG-injected larvae while control larvae reached the premolt peak. These results indicate that a molt-inhibitory mechanism similar to that of juvenile and adult decapod crustaceans may also exist in lobster larvae.

Intraspecific competition and aggression for shells in the hermit crab Pagurus samuel1s

Many studies have investigated shell‐related behaviour in hermit crabs. Few studies, however, have focused specifically on the intraspecies aggression associated with shell competition. We examined intraspecies aggression in hermit crab (Pagurus samuelis) pairs as it relates to competition for a limiting resource, gastropod shells. Pairs of hermit crabs were observed in the laboratory in four different treatments that varied the presence or absence of shells for one or both of the crabs. Measurements of the latency to respond, the number of bouts, and the fight durations were recorded. There was a significant difference among treatments for all three measurements, and naked hermit crabs were much more aggressive than housed hermit crabs. There was no significant difference in aggression between males and females in any of the three treatments. The heightened aggression observed in naked P. samuelis is likely in service of acquiring a protective shell.