Multilevel analyses of endocrine disruption in shore crabs from UK estuarine and coastal environments
Department Contract Reference Number: CDEP/84/5/309
Date of Report: 25 April 2003
Contract start and end dates: 1 April 2001 to 31 December 2001
A.S. Clare, M.G. Bentley & R.J. Ladle
School of Marine Science & Technology, Ridley Building, Newcastle University, Newcastle upon Tyne, NE1 7RU.
Contents Page
Page
List of figures and tables……………………………………………………..………. 3
Executive Summary………………………………………………………………….. 4
Introduction………………………………………………………………………….. 5
Background…………………………………………………………………………... 5
Objectives……………………………………………………………………………. 6
Methods……………………………………………………………………………… 6
i) Collection /sampling sites………………………………. ………………………. 6
ii) Morphometric analyses of Carcinus maenas…………………………………. 7
iii) Behavioural assays of interference in the normal pattern of sex
pheromone communication of Carcinus maenas………………………………. 8
iv) Radioimmunoassay of haemolymph 20-hydroxyecdysone
(20E) titres………………………………………………………………………….. 9
Studies Conducted………………………………………………………………….. 9
Milestone 1; April – June 2001……………………………………………………… 9
Milestone 2; July – September 2001……………………………………….………. 9
Milestone 3; October – December 2001…………………………………………… 9
Milestones met……………………………………………………………………….. 10
Milestones not met…………………………………………………………………… 10
Results and observations…………………………………………………………. 10
i) Collection /sampling sites………………………………. ………………………. 10
ii) Morphometric analyses of Carcinus maenas…………………………………. 10
iii) Behavioural assays of interference in the normal pattern of sex
pheromone communication of Carcinus maenas………………………………. 12
iv) Radioimmunoassay of haemolymph 20-hydroxyecdysone
(20E) titres………………………………………………………………………….. 15
Analysis and discussions………………………………………………………….. 15
Conclusions………………………………………………………………………… 15
Recommendations………………………………………………………………… 15
Contacts with other organisations……………………………………………….. 16
Publications………………………………………………………………………… 16
References…………………………………………………………………………. 16
List of figures and tables
Page
Fig. 1. Top - Female crab showing normal abdominal morphology;
Middle: Crab showing “intersex” abdominal morphology; Bottom:
Male crab showing normal abdominal morphology……………………………… 7
Fig. 2. Normal female (left hand panel) and ‘superfemale’ (right hand panel)
crabs collected from the Tees estuary……………………………………………. 7
Fig. 3 males taken from pre-copula pairs displayed the full sequence of
sexual behaviours culminating in cradling of the stone, which had been
dipped in female-conditioned water (pheromone), in a protective embrace…. 8
Fig. 4 Arisaig male responses to “dipped” stone………………………………… 12
Fig. 5 Oban male responses to “dipped” stone………………………………….. 13
Fig. 6 Lindisfarne male responses to “dipped” stone…………………………… 13
Fig. 7 Tyne male responses to “dipped” stone………………………………….. 14
Fig. 8 Tamar male responses to “dipped” stone………………………………… 14
Table 1 Male claw depth – analyses using GLM………………………………… 11
Table 2 Male abdomen areas – analysis using Mann-Whitney U-test………… 11
Table 3 Male pleopod lengths – analysis using GLM…………………………… 11
Table 4 Female abdomen areas – analyses using GLM………………………… 11
Table 5 Female right pleopod lengths – analyses using 2-sample T test……… 11
Executive Summary
This study was undertaken to examine possible 'sex change' effects (i.e. endocrine disrupting effects) such as interference in mating behaviour and morphology of the shore crab Carcinus maenas. Specimens were collected repeatedly from a number of sites around the UK:
· Tyne, Tees & Tamar, as potentially ‘polluted’ sites
· Oban, Arisaig and Lindisfarne as ‘unpolluted’ sites.
Animal collection was supplemented by collection of sediment and water samples, and invertebrate blood (haemolymph) samples taken from all crabs tested in bioassays and for which morphometric analyses were carried out. These have been archived and will be available for subsequent chemical analyses.
The main points of the study are as follows:
· Morphometric analyses focused on abdominal morphology, claw depth and pleopod leg length.
· Abdominal morphology varied from normal showing ‘intersex’ (male crab which expresses female-like morphological characteristics) or ‘super-female’ (female displaying enlarged abdominal morphology) characteristics.
· Male claw depth of Tamar (‘polluted’ site) crabs was smaller compared to Lindisfarne (‘pristine’ site) crabs.
· Male pleopod leg length did not differ significantly between sites.
· Abdomen area and male claw depth data suggest that further study on these characters is warranted.
· Observation of inter-moult males, males taken from pre-copula pairs and crabs showing ‘intersex’ morphology.
· Male and female shore crabs produce sex pheromones. These are chemical substances that attract the opposite sex prior to the female moult and control mating behaviour. Comparisons of pheromone-induced sexual behaviour were made of crabs from Arisaig, Lindisfarne, Oban, Tamar and Tyne.
· These analyses were undertaken using a ‘stone bioassay’, in which a crab’s response to a pheromone-dipped stone was recorded.
· For all sites there was a strong relationship between ‘intersex’ morphology and a lack of response to pheromone-dipped stones. However, as no ‘intersex’ crabs were found in mating pairs, we could not be certain that the males were responsive to pheromone. Evidence of a feminised response by intersex males in the ‘stone assay’ is, therefore, equivocal.
Introduction to multilevel analyses of endocrine disruption in the shore crabs from UK estuarine and coastal environments.
Background
Evidence for ‘sex effect’ changes or ‘endocrine disruption’ in the aquatic environment is now well founded30, largely with respect to freshwater fish14,16,17,31 and to a lesser extent, estuarine and marine fish1,2,19,20,21. With the exception of the now classical work on TBT and imposex (masculinisation of females) in dogwhelks and other neogastropods22, there have been few comparable investigations of marine invertebrates11. The latter are, however, generally regarded as being more sensitive to contaminants than vertebrates, and there is therefore the potential for effects from exposure to endocrine disrupting chemicals (EDCs). Thus far, studies have largely attempted to determine whether known EDCs have effects on reproduction and development in the laboratory. Here, results that are consistent with endocrine disruption have been noted4,5,6,10,12,18,26,28. Evidence suggestive of endocrine disruption, has also been obtained in the field6,25,27, although it must be emphasised that causal links to EDCs have not been established22. Consequently, there is a clear need to determine whether or not endocrine disruption occurs in the natural environment and thus whether the results of laboratory assays are of ecological relevance. Given that estuaries and inshore waters are subject to most anthropogenic insult (either directly or via river discharge) from e.g. industrial discharges, agricultural run-off and sewage outfalls, the likelihood of obtaining evidence of endocrine disruption in marine invertebrates is highest there. This is largely borne out by the results of field surveys of endocrine disruption in fish1,2,19,20,21.
Matthiessen and colleagues24 have discussed weaknesses in current field monitoring programmes. They also offered guidance on the selection of taxa for monitoring studies, viz. groups should have “1) well characterised endocrine systems wherever possible, 2) ecological importance in ecosystem maintenance, 3) rapid reproductive cycles, 4) sessile or semi-sessile behaviours, 5) widespread distribution, and/or 6) economic importance”. With regard to disruption of reproductive hormones, it would also be advantageous if the species under consideration were known to have the capacity for the intersex (feminisation of males) condition. The common shore crab, Carcinus maenas, fulfils most of these criteria. It does not, however, have a rapid reproductive cycle and is not sessile in its habit. It has been suggested that sessile marine invertebrates are ideal field study organisms24. However, it must be remembered that most sessile marine invertebrates, e.g. barnacles, have planktonic larval stages that can disperse over great distances, and even if endocrine disruption were demonstrated, it would be equally difficult to determine at what point in their life-cycle they had been exposed to EDCs.
In selecting Carcinus maenas as a species for study, we have been mindful that if field studies provide evidence of endocrine disruption, further mechanistic studies to provide, e.g., biomarkers for monitoring require a detailed knowledge of the organism’s endocrinology. Unlike most marine invertebrates in UK waters, however, the endocrinology of decapod crustaceans, such as Carcinus maenas and Cancer pagurus, is comparatively well understood. Moreover, intersex in C. maenas which have been parasitised by the thoracican barnacle Sacculina carcini is well known; effects of parasitisation include castration and cessation of moulting. In males, secondary sex characteristics of the female, including broadening of the abdomen, are induced. The mechanism whereby these effects are induced is not known, but an effect on the endogenous endocrine regime of the crab, through androgen gland degeneration, has been postulated15.
Indications that non-parasitised C. maenas are indeed susceptible to endocrine disruption have been provided by a preliminary unpublished study (Brian et al., in revision). In a survey of three sites - Redcar Jetty (Tees estuary), Tynemouth (north of the Tyne estuary) and Dunstaffnage Bay – differences in morphological characters indicative of inter-sex were detected (Figures 1 and 2). Dunstaffnage Bay was selected as a relatively pristine site for baseline study and indeed no evidence of inter-sex was detected there. The Tyne and the Tees estuaries, on the other hand, have been shown to be impacted by endocrine disrupters1,2,21,23 and inter-sex characters in C. maenas collected at these sites are consistent with endocrine disruption. Further work is now required to validate these findings and to examine further the possibility of using C. maenas as an indicator of endocrine disruption in estuarine and marine environments. The present proposal identifies a multilevel approach to these issues. With regard to the general objectives outlined in the DETR’s research call, the proposal thus focuses on identifying whether or not endocrine disruption is occurring rather than identification of the causes. The latter would be premature with regard to marine invertebrates with the possible exception of imposex in gastropods.
Objectives
The overall approach of this study was to use a combination of morphometric and behavioural analyses to assess possible endocrine disruption in Carcinus maenas.
Three specific objectives were identified:
1) To carry out morphometric measures on field collected crabs to confirm the findings of a preliminary study by Richard Ladle, and to extend the survey to include sites in northwest and southwest England, which have been surveyed previously for endocrine disruption in fish.
3) To investigate if sex pheromone production in ‘super female’ crabs and the behavioural response of inter-sex ‘male’ crabs to female sex pheromone differ from that of ‘morphometrically normal’ crabs.
3.) To investigate if crab steroid moulting hormone titre of 20 hydroxyecdysone of inter-sex and ‘superfemale’ crabs differ from that of morphometrically normal crabs of each sex.
Methods
i. Collection/sampling sites
Collection was proposed to be undertaken from a number of ‘polluted’ sites (Tyne, Tees, Dee, Tamar and Mersey) and ‘unpolluted’ sites (Oban, Arisaig and Lindisfarne) around the UK based on observations made during a pilot study (Brian et al., unpublished). Animal collection was supplemented by collection of sediment and water samples, which have been archived and will be available for subsequent chemical analyses.
ii. Morphometric analyses of Carcinus maenas
Morphometric analyses focusing on secondary sexual characteristics (abdomen area; claw depth and pleopod leg length) were carried out on 30 females and 30 males collected during the course of the study from the sites described above. Analyses were performed with UTHSCSA Image Tool 2.0 and a digital image was recorded for each crab. Categories of abnormalities are illustrated in figures 1 and 2. One set of crabs from an unpolluted site (Lindisfarne) was compared with crabs collected from the polluted sites. All crabs were coded and have been archived at –20oC for future reference.
iii. Behavioural assays of interference in the normal pattern of sex pheromone communication in Carcinus maenas
Behavioural analyses examined pheromonally mediated sexual behaviour. There were 3 components to this aspect of the study:
1) Description of sexual behaviour (this was confined to inter-sex crabs as only 2 superfemales were found at the end of the study)
2) Investigation of responses of inter-moult males, males taken from pre-copula pairs and crabs showing ‘inter-sex’ morphology
3) Comparison of pheromone induced sexual behaviour of crabs from Arisaig, Lindisfarne, Oban, Tamar and Tyne.
Sexual behaviour – preliminary analyses of sexual behaviour led to the development of the stone bioassay (Fig. 3) which was adopted for behavioural characterisation of normal and crabs showing ‘inter-sex’ morphological characters.
For the stone assays a male crab is exposed to a stone dipped in female sex pheromone. A positive result comprises the crab treating the stone as a female, grabbing and holding it in a pre-copulatory guarding position. This is to prevent other male crabs mating with the pseudo-female. When an inter-sex crab is exposed to a stone assay, the observed response is generally either to interact with the pheromone-treated stone without exhibiting the pre-copular behaviour or, in most cases, to show no response. Three replicates bioassays of each crab were performed.
2) and 3) As no ‘inter-sex’ crabs in pre-copula were found, ‘inter-sex’ assays were carried out on singleton crabs. These were compared with males showing normal morphology during intermoult and from pre-copula pairs. In all, over 300 bioassays were carried out.
iv. Radio-immunoassay of haemolymph 20-hydroxyecdysone (20E) titres.
Haemolymph samples were taken from inter-moult males and females, pre-moult females, morphological abnormal males (warped telson) and inter-sex males. In addition, samples of haemolymph were taken from super-females. These samples were intended for radioimmunoassay (RIA) to determine the titre of the arthropod moulting hormone, 20-hydroxyecdysone, since steroid hormones have been shown to be particularly important in endocrine disruption. Haemolymph samples were subsequently alcohol extracted and stored at -80°C.
Studies Conducted
The milestones of the programme were as follows:
Milestone 1; April-June 2001
· All sites have been visited and crab collections made from Tyne, Tees, Tamar (potentially 'polluted' sites) Oban, Arisaig and Lindisfarne ('unpolluted' sites).
· Sediment and water samples collected from all sites and are now stored at -20°C in glass containers for future analyses.
· Crabs have been analysed morphometrically (measurements of both sexually dimorphic and general characteristics). Results of initial morphometric analyses have been stored on CD.