Intermoult Duration Research Articles

Effects of long-term chlorpyrifos exposure on moulting and growth of Armadillo officinalis Dumèril, 1816 (Crustacea, Isopoda, Oniscidea)

The aim of this study was to evaluate the long-term effects of the insecticide chlorpyrifos on the moulting and growth of the terrestrial isopods Armadillo officinalis. Adult woodlice were exposed to chlorpyrifos at concentrations of 5, 10, 20, 40 mg/kg dry soil and compared to a control. During 100 days of exposure, effects on moulting and growth were investigated. The duration of premoult, ecdysis and intermoult stages were estimated. Also, the cuticle width (CW) and length (CL) were measured and their size increase and specific growth were calculated. The results showed that exposure to chlorpyrifos induced adverse effects on moulting in the terrestrial isopod Armadillo officinalis, mainly at the higher concentrations (20 mg and 40 mg/kg dry soil). Indeed, it decreased the percentage of moulting animals, delayed moulting, decreased the number of completed moult, extended the premoult stage, prolonged ecdysis, and reduced intermoult duration. Furthermore, these findings showed that chlorpyrifos exposure impaired also growth of Armadillo officinalis, mainly at the highest concentration (40 mg/kg dry soi), by decreasing weight gain (0.94 ± 2.14 mg), cuticle length gain (0.26 ± 0.06 mm) and specific growth rate (SGR) (0.04 ± 0.01 mm/days) compared to the control (Weight gain = 16.5 ± 1.84 mg; CL gain = 0.65 ± 0.07 mm; SGR = 0.08 ± 0.01). The present study highlighted the adverse effects of chlorpyrifos on moulting and growth of Armadillo officinalis, which raises concern about consequences of chlorpyrifos exposure on population dynamic of this species.

Astaxanthin: The ubiquitous and abundant carotenoid as a pivotal interior factor of anti‐oxidation and immune for the moulting of Portunus trituberculatus

This study was designed to demonstrate the pivotal carotenoid(s) contents and its correlation with anti-oxidative capacities in the moulting of P. trituberculatus. Experiments were carried out to investigate the cyclic fluctuations of carotenoids contents, anti-oxidative indices activities (contents) and non-specific immune indices activities (contents) in the hepatopancreas, haemolymphs and epithelia of juvenile P. trituberculatus during the moulting cycle. Further, a regressing analysis was made on the correlation between carotenoids contents and anti-oxidative indices values during the moulting cycle. The results showed that the total anti-oxidative capacity and immunity (in haemolymphs) at ecdysis decreased drastically, even though other anti-oxidative indices activities increased, when compared to those in the intermoult duration (or premoult) respectively. The decreases in T-AOC mean juveniles suffer excess oxidative damages at ecdysis. Both triglycerides and carotenoids were taken up in hepatopancreas during the inter-moult, then consumed mostly in the moulting process. The carotenoids compositions analysis results not only illustrated astaxanthin was the only carotenoid species exiting ubiquitously in hepatopancreas, haemolymphs and epithelia in all moulting stages, but also showed astaxanthin was the only one detected in haemolymphs in all moulting stages. Following correlation regression results showed astaxanthin contents were positively related to total antioxidant capacity values in haemolymphs. Accordingly, astaxanthin should be an important anti-oxidative factor in haemolymphs during the moulting. In conclusion, those results clearly suggested astaxanthin should be the ubiquitous and abundant carotenoid for anti-oxidation in P. trituberculatus during the moulting cycle. This awareness may provide a understanding of astaxanthin physiological mechanism in P. trituberculatus.

The impact of seismic survey exposure on the righting reflex and moult cycle of Southern Rock Lobster (Jasus edwardsii) puerulus larvae and juveniles

Anthropogenic aquatic noise is recognised as an environmental pollutant with the potential to negatively affect marine organisms. Seismic surveys, used to explore subseafloor oil reserves, are a common source of aquatic noise that have garnered attention due to their intense low frequency inputs and their frequent spatial overlap with coastal fisheries. Commercially important Southern Rock Lobster (Jasus edwardsii) adults have previously shown sensitivity to signals from a single seismic air gun. Here, the sensitivity of J. edwardsii juveniles and puerulus to the signals of a full-scale seismic survey were evaluated to determine if early developmental stages were affected similarly to adults, and the range of impact. To quantify impact, lobster mortality rates, dorsoventral righting reflex and progression through moult cycle were evaluated following exposure. Exposure did not result in mortality in either developmental stage, however, air gun signals caused righting impairment to at least 500 m in lobsters sampled immediately following exposure, as had previously been reported in adults with corresponding sensory system damage following exposure. Impairment resulting from close range (0 m) exposure appeared to be persistent, as previously reported in adults, whereas juveniles exposed at a more distant range (500 m) showed recovery, indicating that exposure at a range of 500 m may not cause lasting impairment to righting. Intermoult duration was (time between moults) significantly increased in juveniles exposed at 0 m from the source, indicating the potential for slowed development, growth, and physiological stress. These results demonstrate that exposure to seismic air gun signals have the potential to negatively impact early life history stages of Southern Rock Lobsters. The similarity of both the impacts and the sound exposure levels observed here compared to previous exposure using a single air gun offer validation for the approach, which opens the potential for accessible field-based experimental work into the impact of seismic surveys on marine invertebrates.

Taxonomic and genetic confirmed findings of snow crab (Chionoecetes opilio) larvae in the Barents Sea

The snow crab (Chionoecetes opilio) is an Arctic cold-water species native to the northwestern Atlantic Ocean and the northern Pacific Ocean. During the recent decades, a population has established in the Barents Sea. Several aspects of the snow crabs’ biology in this area have not been described, including time of hatching, intermoult duration of the different larval stages and larval distribution. Insight into the early-life stages might increase the understanding of the population's dynamics and further spreading in the Barents Sea as well as inform basis for making monitoring and management decisions. The present study investigated the presence and developmental stage of snow crab larva in plankton samples obtained in the central Barents Sea during a research survey in June and July 2019. Presence of snow crab larvae was confirmed through taxonomic and genetic identification. All larvae were identified as zoea I, which gives an indication of the timing of the hatching period. Morphological measurements coincide well with those reported in studies from the species native distribution range. No larvae of native Hyas spp. were found and overlap in temporal and spatial distribution is discussed. The study provides important information for development of further research into the biology of the snow crab in the Barents Sea.

Effects of temperature, fungal infection and weight on intermoult duration and survival of starving earwig larvae

Moulting is a cornerstone of arthropods development. It can be determined by numerous factors such as body mass, temperature, and immunity. However, the effects of these factors can be dependent on each other, so that it is often difficult to predict whether and how they shape moulting, and whether their effects are additive or interactive. In this study, we addressed these questions by testing the effects of body mass, ambient temperature, fungal infection and their interaction on intermoult duration and survival in starved juveniles of the European earwig Forficula auricularia. We recorded the date of moult and death of a total of 207 earwig juveniles that were weighed, exposed to different doses of the entomopathogenic fungus Metarizium brunneum and then maintained at either 20 °C or 24 °C. Our results first reveal that juveniles moulted earlier when they were heavy compared to light on the day of exposure, as well as earlier when maintained at 24 °C compared to 20 °C. By contrast, pathogen exposure did not affect the moulting date. We also found that nymphs died faster when they were light compared to heavy on the day of exposure, when they were exposed to high (106 and 107 spores/ml) compared to low (104, 105 and 0 spores/ml) pathogen concentrations, and when they were maintained at 24 °C compared to 20 °C. We detected no sign of interaction between temperature, fungal infection and body mass on both moulting and survival. Overall, these findings shed light on the limited importance of infection on moulting in starved juveniles, and reveal that weight, temperature, and infection have additive effects on their survival. More generally, this study emphasizes that the three tested factors do not necessarily interact to shape key physiological processes in an insect.

Individual growth pattern of juvenile stages of the Chinese mitten crab (Eriocheir sinensis) reared under laboratory conditions

The specific growth pattern of Chinese mitten crab, Eriocheir sinensis, during the juvenile stages was investigated under an individual rearing system for 160 days, including parent crab selection, hatchery management, larval stages, and juvenile cultivation. There were 36 males and 40 females developed from megalopa to the juvenile crab stage 10 (M–C10), with a total survival of 38%. The survival rate in early stages (M–C3) remained less than 80%, which was lower than that in latter stages (> 90%). The intermoult duration sharply increased in an exponential manner (y = 3.1059e0.2149x R2 = 0.9383) from 4 ± 0.54 days to 38 ± 6.26 days. The increments in wet weight, carapace width, and carapace length per moult were recorded throughout the experiment, which followed certain patterns with progressing moulting time. In addition, moulting increment in wet weight varied greatly from C1 to C6, with a minimum increment of 108.09%. Meanwhile, the specific growth rate markedly increased in the early stages and subsequently decreased, mainly because of significant increases in the intermoult duration starting from C6 stage. Males and females could be differentiated at C4 based on sexual dimorphism in the abdomen, and the most distinct changes in female and male juvenile crabs occurred in the shape of the abdomen and amount of cheliped fluff, respectively.

Decreased pH and increased temperatures affect young-of-the-year red king crab (Paralithodes camtschaticus)

The red king crab (Paralithodes camtschaticus) is a high-latitude commercially important species with a complex life-history cycle which encompasses a wide variety of conditions and habitats. High-latitude waters, including those around Alaska where red king crab live, are predicted to have increased ocean acidification and temperatures in comparison to other areas. The interaction of ocean acidification and increased temperature has not been examined for any life history stage of red king crab. To determine the effects of near-future ocean acidification and warming temperature on young-of-the-year red king crab survival, growth, and morphology, we conducted a long-term (184 d) fully crossed experiment with two pHs and three temperatures: ambient pH (∼7.99), pH 7.8, ambient temperature, ambient +2 °C, and ambient +4 °C, for a total of six treatments. Mortality increased with exposure to reduced pH and higher temperatures, but a clear trend in the interactive effects of the stressors was not observed. A synergetic effect on mortality was observed in the pH 7.8 and ambient +4 °C temperature treatment. This treatment also had the lowest survival with only 3% surviving to the end of the experiment. However, an antagonistic effect on mortality was observed in the pH 7.8 and ambient +2 °C treatment. Lower pH and warmer temperatures affected intermoult duration, only temperature affected percent increase in size, but carapace length was not affected. Decreased pH and increased temperature had no effect on morphology. The results of this study combined with other studies show that decreased pH and warming has profound negative effects on red king crab. Unless the species is able to adapt or acclimate to changing climate conditions, red king crabs populations may decrease in the upcoming decades due to ocean acidification and rising temperatures.

Growth and reproduction investment of the young of the year of the squat lobster <em>Munida gregaria</em> (Crustacea: Anomura) in the Patagonian coast

The growth and reproduction investment of the young of the year (0+) of the squat lobster Munida gregaria in San Jorge Gulf, Argentina, were analysed. Moult cycle and size frequency distribution were studied in monthly field samples. Experiments to determine moult increments and intermoult duration were conducted. Sexual maturity, fecundity and reproductive output were analysed in field samples. In addition, maturity experiments in laboratory cultures were conducted. After settlement (November), juveniles showed fast growth until winter (July) and growth restarted in September-October. At the beginning of the mating period (early June), males 0+ exhibited mature gonads, whereas females 0+ only showed previtellogenic oocytes. Experimental data showed that females with developed gonads had smaller-size increments in the previous moult. Fecundity and reproductive output were significantly lower in females 0+ than in older females. The early life history of M. gregaria in San Jorge Gulf differs from that of southern populations as those of Beagle Channel and Strait of Magellan due to faster growth and early reproductive investment. Our results suggest that females 0+ of M. gregaria have two investment strategies: early maturity with low fecundity and delayed maturity with higher future fecundity.

Individual and mixture effects of selected pharmaceuticals on larval development of the estuarine shrimp Palaemon longirostris

Few ecotoxicological studies incorporate within the experimental design environmental variability and mixture effects when assessing the impact of pollutants on organisms. We have studied the combined effects of selected pharmaceutical compounds and environmental variability in terms of salinity and temperature on survival, development and body mass of larvae of the estuarine shrimp Palaemon longirostris. Drug residues found in coastal waters occur as mixture, and the evaluation of combined effects of simultaneously occurring compounds is indispensable for their environmental risk assessment. All larval stages of P. longirostris were exposed to the nonsteroidal anti-inflammatory drug (NSAID) diclofenac sodium (DS: 40 and 750μgL−1), the lipid regulator clofibric acid (CA: 17 and 361μgL−1) and the fungicide clotrimazole (CLZ: 0.14 and 4μgL−1). We observed no effect on larval survival of P. longirostris with the tested pharmaceuticals. However, and in contrast to previous studies on larvae of the related marine species Palaemon serratus, CA affected development through an increase in intermoult duration and reduced growth without affecting larval body mass. These developmental effects in P. longirostris larvae were similar to those observed in the mixture of DS and CA confirming the toxic effects of CA. In the case of CLZ, its effects were similar to those observed previously in P. serratus: high doses affected development altering intermoult duration, tended to reduce the number of larval instars and decreased significantly the growth rate. This study suggests that an inter-specific life histories approach should be taken into account to assess the effect of emergent compounds in coastal waters.

Environmental effects on fished lobsters and crabs

The fisheries for crabs and lobsters (Reptantia, Decapoda) are shaped by environmental variation through the distribution ecology, productivity or even their market traits such as colour and size. Many crabs and lobsters have a wide latitudinal distribution and therefore are exposed to significant abiotic gradients throughout their geographic range. Environmental factors affect reptantians throughout their complex life cycle, including embryo development, timing and length of the spawning period, the duration and quality of the larval stages, the level and spatial distribution of the settlement, growth rates and size of the juveniles, size at maturity, and catchability. The most consistent environmental response is of growth and reproduction to temperature. Growth rates increase with increasing temperatures in a parabolic function, tapering and then declining as the boundaries of thermal tolerance are reached. With increasing temperature the intermoult duration decreases. Once the upper thermal boundary is reached, increases in temperature result in longer intermoult duration and smaller growth increments so that growth is reduced. Declines in temperature generally suppress moulting, and consequently reptantians rarely moult in winter. Increasing temperature decreases the time for egg incubation, larval development and the maturational age. Catchability increases with water temperature and also varies, although less predictably, with moon phase and wind strength. Catchability decreases with an increase in population density. Larval settlement of many reptantian species depends on current strength, increasing with the strength of certain local currents. Reptantians can tolerate a wide-variety of conditions and have flexible life-histories to respond to conditions throughout their broad geographic ranges. Information on environmental effects on reptantians not only assists in understanding probable effects of ocean warming and acidification, but also seasonal and interannual changes in fisheries production.

A multiple stressor approach to study the toxicity and sub-lethal effects of pharmaceutical compounds on the larval development of a marine invertebrate

We studied the effects of three common pharmaceutical compounds on growth, development and body mass of larval stages of the marine shrimp Palaemon serratus at different temperatures and salinities. The pharmaceuticals compounds tested were the anti-inflammatory and analgesic diclofenac sodium, the lipid regulator clofibric acid and the fungicide clotrimazole. Neither diclofenac nor clofibric acid had any effect on growth, development or survival, although the maximum concentrations tested were 40 times higher than those observed in European coastal waters. Clotrimazole had significant effects at the higher concentration (2.78μgL−1) when larvae were reared in full salinity sea water (32PSU) and at the lower concentration (0.14μgL−1) when larvae were reared at 20PSU. Changes in body mass at larval stage resulted from effects of these compounds on growth and developmental rates, specifically the changes in intermoult duration and in the number of larval instars required to reach the juvenile stage. The results demonstrate that the effects of emergent compounds on growth and development may be stronger when organisms are under some additional stress.

Differential coloration patterns in the intertidal crab Neohelice (=Chasmagnathus) granulata

Many crustaceans with spatial segregation may present differential body coloration depending on the habitat they inhabit. Since crustaceans are unable to synthesize carotenoids, individuals must acquire them from their diet, so diet changes can modify individual coloration. The burrowing crab Neohelice (=Chasmagnathus) granulata inhabits the intertidal, from the uppermost parts of salt marshes to the lowest mudflat zones with a spatial segregation of different size-classes. They are primarily deposit feeders in mud flats and herbivorous–detritivorous in the salt marsh. We analysed the effect of intermoult length on the carapace colour of N. granulata, and the changes in carapace colour with crab size and zone. Crab colour varied between unvegetated and vegetated areas. Some of these differences were in part caused by colour changes related to crab size. Larger crabs show less intensive colours, and given that they inhabit the upper part of the intertidal a differential coloration pattern occurred between zones. In field experiments intermoult duration affected crab colour and some body regions analysed showed differences between zones unrelated to size.

Developmental strategies in an invasive spider: constraints and plasticity

1. Growth defines the major life-history traits such as size, weight, and age at maturity that determine an organism's fitness. Different models have been developed to describe growth by means of geometric progressions (e.g. Dyar's rule). However, growth forced along a geometric trajectory might constrain a plastic response to variable environmental conditions (e.g. food availability). 2. The present study investigated growth patterns under varying food conditions in the bridge spider, Larinioides sclopetarius, an extremely successful species in colonising urban habitats. 3. In L. sclopetarius growth ratios of successive instars were not constant but decreased over development. Instead, these spiders' growth is well described by a developmental growth rate (weight gain per moult) and a growth coefficient (weight gain per development time), both of which are based on a geometric progression. All developmental parameters, including developmental growth rate and growth coefficient as well as the intermoult duration and the number of instars, highly depend on food availability in L. sclopetarius and thus show plasticity. 4. Our study shows that geometric growth patterns do not necessarily preclude plasticity and that the parameters of geometric growth are affected by developmental plasticity. We suggest that their high developmental plasticity may facilitate bridge spiders' success in invading urban habitats.

Does foreplay matter? Gammarus pulex females may benefit from long-lasting precopulatory mate guarding

Precopulatory mate guarding (PCMG) is generally assumed to be costly for both sexes. However, males may gain by displaying long-lasting mate guarding under strong male-male competition. Surprisingly, the potential for females to benefit from being held by males has been largely overlooked in previous studies. In Gammarus pulex, an amphipod crustacean, PCMG lasts several weeks, yet females are described as bearing only cost from such male mating strategy. We investigated potential female benefits by assessing the effect of mate guarding on her intermoult duration. Unpaired females had longer intermoult duration than paired females. Intermoult duration clearly decreased when paired females engaged in early and long-lasting mate guarding. In addition, short intermoults and long-lasting mate guarding had no effect on egg number. These results highlight a potential benefit associated with PCMG for G. pulex females, suggesting that the strength of an intersexual conflict over its duration may be overestimated.

Development and growth of the early juveniles of the spider crab Maja squinado (Brachyura: Majoidea) in an individual culture system

The spider crab Maja squinado is an endangered Mediterranean species; therefore, culturing it successfully is essential for developing restocking programs. The survival, growth and development of post-larval stages (juvenile crabs, C1–C8) were studied using larvae obtained from adult individuals collected in the Catalan Sea. The juvenile crab stages were cultured individually from a megalopal stage using a semi-open recirculation system to obtain the precise growth data of each juvenile crab stage until C8. Development up to C8 at 20 °C lasted 154 ± 10 days. Survival from C1 to C8 was 5.8%. Moult increment values in cephothoracic length were similar in all the crab stages (21–35%). Intermoult duration (9 ± 1 in C1–C2 to 51 ± 8 days in C7–C8) increased sharply from juvenile stage 5. Males and females can be distinguished from C4 based on sexual dimorphism in the pleopods and the presence of gonopores. The allometric growth of the pleon is sex-dependent from C4, with females showing positive allometry and males isometric growth. The juvenile growth rate was lower compared with that of the previously studied Atlantic species Maja brachydactyla.

Dietary protein requirement of giant mud crabScylla serratajuveniles fed iso-energetic formulated diets having graded protein levels

The protein requirement of juvenile mud crab Scylla serrata (body weight=0.25±0.051 g, carapace width=9.3±0.04 mm) fed with different iso-energetic, iso-lipidic diets with graded protein levels (15–55% crude protein at 5% intervals) was determined. The feeding trial was conducted for a period of 63 days to determine the minimum and optimum protein requirement of juvenile S. serrata. The crabs fed with 15% and 20% dietary protein levels showed 100% and 12.5% of mortalities respectively. The mortalities observed in the above treatments were associated with the prolonged intermoult duration (46 and 32 days respectively). All other treatments recorded 100% survival. The best growth performance as well as the nutrient turn-over was recorded in crabs fed with 45% crude protein in the diet. Second-order polynomial regression of specific growth rate (SGR) as well as body protein gain vs. dietary protein levels suggested that 46.9–47.03% dietary protein is required for the best growth response and protein deposition in juvenile S. serrata. An extrapolation of ‘SGR’ and ‘daily protein gain’ upon the ‘dietary protein level’ axis (Y=0) showed that 14.7–16.2% dietary protein is necessary for the minimum maintenance metabolism.

Survival and growth of post-settlement juveniles of the spider crab Maja brachydactyla (Brachyura: Majoidea) reared under individual culture system

The effect of an individual culture system on survival and growth of juvenile crab stages 1–8 of the majid crab Maja brachydactyla Balss, 1922 was studied using larvae obtained from adult individuals collected in the NE Atlantic. The juvenile stages were cultured individually from a megalopal stage using a semi-open recirculation system. Forty-eight percent of the megalopae moulted to first juvenile stage. Detailed growth data have been obtained for each juvenile stage. Juvenile development up to juvenile crab stage 8 (C1–C8) at 20 °C lasted 117.5 ± 7.5 days. Survival was high during the whole experiment: 43.75% of C1 survived to perform the moult to C8. Moult increment values (%CL) were similar in all the juvenile stages (27.90–37.04%). Intermoult duration (9.37 ± 1.04 to 33.5 ± 2.64 days) increased sharply from stage 6, and the specific growth rate (SGR) diminished due to the significant increase of the intermoult period.

Effect of autotomy and regeneration of the chelipeds on growth and development in Petrolisthes laevigatus (Guérin, 1835) (Decapoda, Anomura, Porcellanidae)

The capacity to discard and regenerate limbs under conditions of vital risk does increase the probability of survival. Eventually, however, this can negatively influence the performance of other aspects of fitness in the organism affected. The influence of autotomy and regeneration of one or both chelipeds on body growth and intermoult period in Petrolisthes laevigatus was studied; individuals in a broad range of sizes were captured and subjected to autotomy treatments in one or both chelipeds, contrasted against a control group without autotomy. Body growth was measured as the increment of the carapace width after a moulting event. At the same time, the duration of the intermoult was considered as the period of time between two succesive moults. Significantly higher values of response in body growth, intermoult duration, and growth rate were observed in the control group with respect to the group with autotomy treatments. In the growth responses, the phenomenon of suboptimal foraging can be discarded and therefore the more probable explanation for growth decrease is related to reduction of the intermoult period or for changes in the allocation of energy from overall growth processes into the limb regeneration process.

The effect of predator/prey density and water dynamics on feed intake and growth in spiny lobster larvae ( Jasus edwardsii)

Consumption of Artemia by phyllosoma of the spiny lobster Jasus edwardsii was examined under static and turbulent conditions. Phyllosoma larvae were stocked at two densities (2 or 4 phyllosoma container − 1 ; 2P or 4P) and fed juvenile Artemia (1.6 mm total length) at two feed rates (1.5 or 0.75 Artemia ml − 1 ; 1.5A or 0.75A) in 50 ml of seawater. This provided a combination of 4 treatments (2P/1.5A, 2P/0.75A, 4P/1.5A, 4P/0.75A). Daily intake of Artemia by phyllosoma was monitored and assessed relative to moult size and intermoult duration at differing predator–prey densities. Phyllosoma numbers were held constant; in the event of mortality, animals were replaced with others from the same cohort cultured under similar conditions. Phyllosoma endogenous reserves at hatch combined with the lowest ration of 0.75 A were sufficient to ensure normal growth and intermoult duration until Stage II. When phyllosoma were fed for an extended period (several moults), low ration and high phyllosoma density resulted in smaller size and extended intermoult duration. Fluctuations in the feeding pattern of phyllosoma were evident between treatments during the experiment. Phyllosoma with access to more Artemia consumed more, and consumption was reduced prior to a moult, even during early stages of development. The second phase of the study examined the intake of 1.7 mm juvenile Artemia by phyllosoma and their response to flow-induced turbulence with a combination of two water exchange rates (2.5 or 5 times h − 1 ) and two inlet positions (2 cm above the water surface or 0.5 cm above the culture vessel bottom and parallel to it). Larvae exposed to slow flow were larger and had a shorter intermoult duration associated with an increased ability to capture and consume more Artemia. Similar numbers of Artemia were consumed during the intermoult in high flow treatments compared to the low flow, albeit over a longer duration. Phyllosoma development between moults required less Artemia to be consumed under static conditions.

Intermoult duration affects the susceptibility of shore crabs Carcinus maenas (L.) to pyrene and their ability to metabolise it

We have studied pyrene (PYR) toxicity and the ability to metabolise and eliminate PYR in two colour forms of shore crabs Carcinus maenas (Linnaeus, 1758). In addition, we analysed differences in the expression of expressed sequence tags (ESTs) encoding specific cytochrome P450s (CYPs) by quantitative realtime PCR. Green and red intermoult crabs are considered to represent different adaptational life stages, allocating energy into growth (green) and reproduction (red), respectively. PYR injection resulted in significantly higher mortality in red crabs than in green crabs during a 51 days period. PYR is an ideal model PAH compound as only 1 phase I metabolite, 1-hydroxypyrene (1-HP) is formed, which is further conjugated to form various phase II metabolites. In this study, 1-HP was detected only after deconjugation of total PYR derived metabolites indicating that PYR hydroxylation (putatively CYP catalysed) conceivably is the rate-limiting step in PYR metabolism. Investigation of the accumulation of PYR and 1-HP (after deconjugation), in different tissues revealed a significantly higher accumulation of PYR in muscle and epidermis of red crabs compared to green crabs. Consistent with this observation, green crabs had significantly higher levels of 1-HP in the hepatopancreas than red crabs. This indicates that a larger portion of the injected PYR was metabolised into 1-HP in green crabs compared to red crabs. 1-HP was mainly detected in the hepatopancreas confirming its major role in the biotransformation of lipophilic compounds. CYP enzymes typically mediate the phase I hydroxylations of lipohilic contaminants such as PYR. In agreement with the higher rate of conversion of PYR into 1-HP in green compared to red crabs, increased abundance of several CYP transcripts was observed in green crabs. Furthermore, in vitro pyrene hydroxylase assays revealed significantly higher NADPH-depedent pyrene hydroxylase activity in hepatopancreas microsomes of green crabs (18.4 ρmol min −1 mg −1 protein) compared to red crabs (8 ρmol min −1 mg −1 protein). The present study demonstrates that the susceptibility of shore crabs to PYR and their ability to metabolise it is life stage dependent, conceivably due to life stage related differences in the expression of certain CYP genes, suggesting a mechanistic explanation of the observed life stage differences in PYR toxicity.