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Pathological and physiological effects of nicking on brown crab (Cancer pagurus) in the Irish crustacean fishery
Journal of Invertebrate Pathology 112 (2013) 49–56
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Pathological and physiological effects of nicking on brown crab (Cancer pagurus) in the Irish crustacean fishery Jennifer E. Welsh, Pauline A. King, Eugene MacCarthy ⇑ Galway-Mayo Institute of Technology, Galway, Ireland
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Article history: Received 2 January 2012 Accepted 15 August 2012 Available online 24 August 2012 Keywords: Brown crab (Cancer pagurus) Nicking Necrosis Pathology Handling Storage
a b s t r a c t Nicking is used in fisheries to immobilize claws of brown crab (Cancer pagurus) in order to prevent cannibalism and fighting during storage. Nicking fractures the apodemes creating an open wound and damage to the internal claw tissues, which is the most valuable product of brown crab. In turn, this results in a reduction of quality of product and possibly compromises the host’s defence mechanisms to other physiological challenges experienced throughout the post-harvest process. This study assessed the effects of nicking on the physiology and pathology of brown crab from the Irish fishery over 7 days. Results showed significantly elevated levels of muscle necrosis (P = 0.005), total pathologies (P = 0.022) and encirculating granulocytes in nicked crab compared to non-nicked crab. Mean glucose (212.0 lg/mL ± 108.4), lactate (36.52 lg/mL ± 38.74) and RI (11.05n ± 1.78) levels were higher in nicked crab indicating increased stress levels. Overall, histology results showed a significantly higher (P = 0.022) occurrence of pathologies, such as melanised nodules, in nicked animals. In addition to an observed reduction in the quality of claw muscle, nicked crab also showed significantly higher (P = 0.005) levels of necrosis in claw muscle. From the results of this study it is recommended that alternative retention methods are used. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Nicking is the typical method of immobilizing claws (chelae) of brown crab (Cancer pagurus) in Ireland. Nicking renders the claw non-functional by fracturing the apodemes (tendon). It is employed by fishers to prevent damage whilst the animals are grouped and for ease of processing (Newman and Ward, 1973; Jacklin and Combes, 2007; Barrento et al., 2008). Two main techniques are used: ‘French’ nicking, cuts the apodemes between the upper and lower dactyl (Haefner, 1971) while ‘English’ nicking, cuts the apodemes between the palm and pincer (Jacklin, 1998). Nicking is not commonly used in the fishery of other crab or lobster species but is typically substituted by banding the claws. The sloped morphology of brown crab claws means that, within the fishery, banding is not practical. Nicking has been shown to create wounds, damage tissue, alter molting and haemolymph clotting processes, and cause swelling (Beaven and Truitt, 1940; Haefner, 1971; Jacklin, 1998; Jacklin and Combes, 2007). Aerial exposure and other post-harvest processes may reduce the haemolymph clotting capacity (Jacklin, 1998; Jacklin and Combes, 2007). Therefore, Jacklin and Combes (2007) suggested that animals should be placed immediately into seawater after nicking to encourage haemolymph coagulation. ⇑ Corresponding author. Present address: Marine and Freshwater Research Centre (MFRC), Galway-Mayo Institute of Technology, Dublin Road, Galway, Ireland. E-mail address: [email protected] (E. MacCarthy). 0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2012.08.006
However, extended coagulation periods may proliferate the time for which bacterial infections can manifest within the claw tissue, increasing the risk of infection by opportunistic pathogens (Jacklin and Combes, 2007). In addition, ‘English’ nicking results in increased bleeding (Jacklin and Combes, 2007). The claw contains the most valuable product of brown crab, therefore, any damage or deterioration caused to the claw muscle results in a lower quality product. Several studies have documented the transport chain from time of capture for live shellfish from capture to consumer (Hearn, 2005; Uglow et al., 2005; Barrento et al., 2008), where total holding and transport times for some crab species vary between 2 and 14 days (Hearn, 2005; Uglow et al., 2005). In general, crabs are nicked on board the fishing vessel at the point of capture and are subjected to a variety of handling techniques and stressors (Paterson and Spanoghe, 1997; Lorenzon et al., 2008). Post capture stressors include aerial exposure, emersion, vibrations, and environmental (temperature and chemical) changes (Spicer et al., 1990; Paterson and Spanoghe, 1997; Taylor et al., 1997; Lorenzon et al., 2008). Damage has, however, been shown to be dependent on the size, age, diet and reproductive state of the individuals (Kruse et al., 1994). Stressors from post-harvest processes may ultimately result in increased prevalence of infections and mortalities. Current industry practices does not require for mortalities to be documented on a daily basis during transportation and storage, however several studies have observed varying degrees of mortality throughout the market chain (Uglow et al., 1986; Stentiford et al., 2002; Stentiford and Sheilds, 2005; Jacklin and Combes,
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2007; Barrento et al., 2008, 2010). Whilst no publications have noted a direct link between nicking and mortalities, it is hypothesised that such wounds inflicted on the crab may contribute to a reduction in overall fitness of the animals. In recent years physiological parameters have been used to assess stress levels in shellfish during the live marketing chain (Söderhäll and Smith, 1986; Taylor et al., 1997; Paterson and Spanoghe, 1997; Fotedar et al., 2006; Gornik et al., 2008; Lorenzon et al., 2008; Barrento et al., 2010). Haemolymph glucose, lactate (used to indicate haemolymph protein concentration) and refractive index have proven to be efficient parameters in measuring crustacean stress levels (Durand et al., 2000; Bergmann et al., 2001; Jussila et al., 2001; Speed et al., 2001; Wyman et al., 2005; Lorenzon et al., 2007). Previously, blood refractive index has been used as a fast response detection method for determining the nutritional condition of decapods (Oliver et al., 2001) due to its correlation with protein concentrations and muscle tissue (Stewart and Li, 1969; Moore et al., 2000). Barrento et al. (2010) noted that monitoring stress responses identifies areas for improvement and may result in the development of enhanced procedures, and therefore, a reduction in mortality rates. Furthermore, quantification of hyaline and granulocyte cells in the form of total or differential haemocyte counts have been used to gauge the health of crustaceans (Smith and Chisholm, 1992; Lorenzon et al., 2000, 2007, 2008). Semi-granulocytes and granulocytes store prophenoloxidase, which initiates pro-PO system, an in vitro phagocytosis reaction (Johansson and Söderhäll, 1985; Söderhäll and Aspan, 1993). During the pro-PO system granulocytes encapsulate foreign material, including bacteria and other pathogens, resulting in melanisation of the tissue (Smith and Söderhäll, 1983; Hose and Martin, 1989; Battistella et al., 1996). After encapsulation both granulocytes and semi-granulocytes initiate melanin synthesis (Smith and Söderhäll, 1983) which breaks down the foreign object. Semi-granulocyte cells respond specifically to polysaccharides found in microbes (Johansson and Söderhäll, 1985). In addition to physiological parameters histological methods can be imperative in assessing tissue degradation. Histology has previously been used to evaluate the effects of post-harvest processes on crustaceans (Paterson et al., 1997; Stentiford and Neil, 2000; Ridgway et al., 2006) and is particularly valuable when assessing muscle necrosis. Necrosis may be a result of damage caused by post capture handling and potentially lead to mortalities further down the marketing chain (Stentiford and Neil, 2000). Finally, brown crab are commercially important throughout Europe and are considered to be the most commercially valuable shellfish species within Ireland (Tully et al., 2006) with landings of ca. 6284 tonnes, worth €13,228,812 in 2008 (Anon, 2009). Little consideration has been previously given to the effects of nicking as a point of entry for opportunistic bacteria and potential pathogens. In keeping with commercial practices, the pathological and physiological effects of nicking on brown crab in the Irish fishery were assessed over a 7 day period. 2. Materials and methods 2.1. Experimental design Two hundred and seventy female brown crab (550–800 g) were caught off Malin Head, Co. Donegal, Ireland. Of these, 135 crabs were nicked using the commercial French method and 135 were banded using 5–10 cm inner tube lengths. Ten nicked crab and 10 non-nicked were sampled on landing (day 0) and the tissue stored for histological analysis (as described in Section 2.2). In addition, haemolymph samples were obtained (as described in Section 2.3) from five non-nicked and five nicked animals for glucose and lactate analysis.
Remaining crabs were transported by road in fish boxes to Clarinbridge, County Galway (ca. 331 km; 5 h) at 6 °C. On arrival both the non-nicked and nicked crab were placed in four 200 L tanks with shared re-circulated natural seawater. Transportation and holding methods utilised in this study were similar to the methods and timescale employed by the Irish crab fishery. During this storage period crab were not fed and the water quality was monitored throughout the trial. Parameters measured included temperature, salinity, pH, Nitrate, Nitrite and Ammonia using Hach Lang DR 800 colorimeter. In addition, mortalities, limb loss and general crab condition were recorded. 2.2. Histology Following anesthetising on ice 10 nicked crab were dissected at 0, 1, 2, 3, 4, 5 and 7 days post nicking, with 10 corresponding controls (non-nicked crab) also dissected per sampling point. Approximately 7 mm2 samples of heart, hepatopancreas, gill, gut, gonad, claw muscle and epidermis were placed into Davidsons seawater fixative (Hopwood, 1996; Howard et al., 2007) for 24 h, after which they were stored in 70% ethanol pending further investigation. Due to the large number of crabs and the variety of methods being used for this study, the realistic number of tissues per crab that could be sampled was limited to seven. However, the sampled tissues were chosen because they provide a good overview of the animal’s health status. Processing was undertaken using standard histological procedures (Feist et al., 2004). Following embedding, 3–5 lm sections were mounted on slides and stained using the Hematoxylin and Eosin Y (H&E) stain method as in Johnson (1980). The tissues were then examined using a light microscope (Olympus BX41) and camera system (Olympus E330 and Olympus Cell-a software). 2.3. Haemolymph D-glucose and L-lactate analysis Haemolymph was extracted (0.2 mL) using a 19 gauge syringe from the basis between the ischium and coax from five nicked and five non-nicked crab on day 0, 2, 4 and 7. Crab subjected to haemolymph extraction were subsequently removed from experimental tanks. Extracted haemolymph was deproteinated by mixing with equal volumes of ice-chilled 0.6 M perchloric acid (Aldrich) and stored at 20 °C until required. Prior to analysis deproteinated samples were defrosted and centrifuged at 8000g for 3 min and the supernatant removed and collected in microtubes. The supernatant of each sample was neutralised by adding 7 lL of potassium bicarbonate (2 M) and gently shaken for 1 min. To complete neutralisation the supernatant was allowed to stand for 5 min at 4 °C. Once neutralisation was complete the precipitated potassium perchlorate formed was separated by centrifugation at 8000g for 1 min and the supernatant collected in microtubes. Quantification of haemolymph D-glucose was carried out by diluting 50 lL of the supernatant with 100 lL of deionised water. 100 lL of the diluted supernatant was transferred to a 96 well plate (Costar). A standard curve was constructed with D-glucose (Sigma) serially diluted (0–80 lg/mL) in deionised water. 200 lL of the assay reagent (0.8 mL of 15.75 mM o-Dianisidine dihydrochloride was added to 39.2 mL of Glucose Oxidase/Peroxidase Reagent (Sigma)) was applied to each well and incubated at 37 °C for 30 min. The reaction was stopped by the addition of 200 lL of 12 N Sulphuric acid and the product absorbance read at 540 nm (Labsystems Multiskan RC). The absorbance values of the standard curve were entered into the program Graph Pad Prisim 4 (GraphPad Software) and the D-glucose concentrations of individual haemolymph samples determined.
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Circulating haemolymph L-lactate levels were measured by taking 2.5 lL of the diluted deproteinated supernatant as outlined above and placed in individual wells of a 96-well plate (Costar). A standard curve was constructed with L-lactate (Trinity Biotech) serially diluted (0–40 lg/mL). 250 lL of L-lactate reagent (Trinity Biotech) was applied to each well and incubated for 5 min at 25 °C. Product absorbance was read at 540 nm (Labsystems Multiskan RC). As outlined for D-glucose, the L-lactate concentrations were determined using the program Graph Pad Prisim 4. 2.3.1. Refractive index Haemolymph was extracted (0.2 mL) using a 19 gauge syringe and placed directly onto the refractometer (E-Line ATC refractometer, Bellingham & Stanley Ltd., UK) and read to assess condition. The refractometer was blanked using deionised water prior to haemolymph analysis. Fig. 1. Percentage cumulative mortality rates over the duration of the experiment (7 days) in both nicked (solid line) and non-nicked (dashed line) crab.
2.3.2. Haemocyte counts Separate samples (0.2 mL) of haemolymph from those taken for the assays were extracted using a 19 gauge syringe and placed directly into 4% neutral buffered formalin (NBF) for fixation. Fixed haemolymph was smeared onto slides and analysed for granulocyte counts using a light microscope (Olympus BX41) and camera system (Olympus E330 and Olympus Cell-a programme). For the purpose of this study, semi-granulocytes were counted as granulocytes. 2.4. Statistics Descriptive statistics using Minitab 15 were carried out on all the data. After testing for normality, parametric and transformed non-parametric data was subjected to an analysis of variance with Tukey’s family error rate. Results that could not be transformed to parametric data was subjected to Wilcoxon signed ranking tests. 3. Results Water quality parameters are shown in Table 1. Temperature ranged from 12.80 °C to 14.90 °C; salinity from 31.00 ppt to 32.50 ppt; pH from 6.23 to 7.10; Nitrate from 5.50.0 mg/L to 24.30 mg/L; Nitrite from 9.00 mg/L to 67.00 mg/L and Ammonia from 0.64 mg/L to 0.73 mg/L. Fig. 1 shows cumulative mortality rates throughout the investigation. Up to day 5, mortality rates were identical in both nicked and non-nicked animals, with an increase in mortality rates observed on day 7 in nicked crab (Fig. 1). Other observations made throughout the duration of the experiment include blackening of the claw tissue around the open wound in nicked crab Fig. 2. This was documented in up to 80% of the nicked crab after day 5, where the extent to which internal tissue is exposed to external factors (Fig. 2C and D). Though not significant, claw loss was greater in nicked crab (mean = 1.43 ± 1.21) compared to non-nicked crab (mean = 0.29 ± 0.49).
3.1. Histology The number of pathologies observed over time in nicked and non-nicked crab is shown in Fig. 3. Both nicked and non-nicked crab showed pathologies throughout the trial. Pathologies observed included granulomas, haemocyte aggregates, necrosis and gill biofilms. The latter are presented in Fig. 4. Pathology data was non-parametric and was therefore subjected to Wilcoxon signed rank tests. Analysis showed a significant difference in pathologies between nicked and non-nicked crab (P = 0.022) over the seven day trial period, with more pathologies observed in nicked crab (collective mean = 15.00 ± 4.24) than non-nicked crab (collective mean = 8.30 ± 2.33) (Fig. 3). In nicked crab the greatest difference was seen between day 0 (mean = 4.50 ± 0.00) and day 4 (mean = 17.30 ± 29.96). However, one animal on day 4 resulted in an outlier with 97 pathologies (Fig. 3). This anomaly showed numerous areas of phagocytises at varying stages of melanisation (from granuloma to melanised nodules) within the connective tissue surrounding the gut. Non-nicked crab recorded the greatest difference in observed pathologies between day 0 (mean = 1.50 ± 0.00) and day 7 (mean = 9.00 ± 1.75). Interestingly, as shown in Fig. 5, non-nicked crab showed a consistent increase in mean number of pathologies unlike nicked crab which showed a more erratic pattern over the 7 days. Necrosis was significantly (P = 0.005) and consistently higher in the claw muscle of nicked crab compared to non-nicked crab (Fig. 5). Over twice the amount of melanisation occurred in the hepatopancreas of nicked crab than in non-nicked crab (mean = 0.19 ± 0.16; mean 0.06 ± 0.06 respectively). In addition, some nicked crab showed haemocyte infiltrations between fibre bundles. Other gross observations showed an increase in hepatopancreatic tubule lumen size and a reduction in R and B cells. Most
Table 1 Water quality parameter measurements taken over the experimental period. Day
Temp. (°C)
Salinity (ppt)
pH
Nitrate (mg/L)
Nitrite (mg/L)
Ammonia (mg/L)
0 1 2 3 4 5 7
12.80 12.80 12.80 12.70 12.70 12.70 14.90
32.50 32.50 32.00 31.30 31.40 31.00 31.10
7.10 7.10 7.06 7.05 7.10 7.01 6.23
550 5.50 20.60 24.30 23.00 24.30 24.30
29.00 29.00 67.00 23.00 47.00 9.00 9.00
0.64 0.64 0.73 0.73 0.73 0.73 0.73
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Fig. 2. (A) The non-nicked tendon between the upper and lower dactyl (arrow) showing no tissue damage (B; arrow); (C) The point where the tendon is fractured (arrow) during French nicking; (D) The blackening of muscle within the claw (arrow). All crabs photographed were from day 7.
Fig. 3. The mean number (±standard error) of pathologies identified in the sampled tissues from nicked (solid line) and non-nicked (dashed line) crabs.
samples showed normal levels of reserve inclusion cells and mature oocytes. 3.2. Haemolymph analysis There was no significant difference between glucose, lactate or RI values in nicked crab compared to non-nicked crab (Fig. 6). However, mean values were higher in nicked crab. Nicked crab showed mean values of 212.0 lg/mL (±108.4), 36.52 lg/mL
(±38.74) and 11.05n (±1.78) for glucose, lactate and RI respectively (Fig. 6). Glucose values for non-nicked crab significantly increased (P = 0.001) over the trial period from 133.60 lg/mL (±39.41) to 303.40 lg/mL (±62.36). Nicked crab glucose values declined from 243.17 lg/mL (±99.37) on day 0 to 151.70 lg/mL (±31.19) on day 7 (Fig. 6). Initial lactate values in nicked and non-nicked crab on day 0 were 89.51 lg/mL (±29.86) and 59.02 lg/mL (±28.56) respectively, decreasing to 6.33.05 lg/mL (±2.42) and 2.50 lg/mL (±0.89) on day 7 (Fig. 6). Post hock tests showed a highly significant difference (P = 0.001) between days 0 and 7 in non-nicked but not in nicked crab. Refractive index (RI) values for nicked crab significantly (P = 0.002) decreased between day 0 (11.46 ± 1.99) and day 7 (9.76 ± 0.96). Non-nicked crab refractive index values did not significantly increase over the trial period, instead mean values fluctuated from 11.62n (±1.25) on day 0 to 10.78n (±0.57) on day 7 (Fig. 6). Changes in encirculating granulocyte numbers were investigated in both nicked and non-nicked crab (Table 2). Granulocytes in non-nicked crab were comparable to levels in non-nicked crab (32.90 ± 12.97 and 32.10 ± 5.65 respectively) at time point 0 (Table 2). An initial increase in granulocyte numbers was observed on day 1 in both groups of crab and this slight increase was maintained through the remaining time points in both nicked (P = 0.032) and non-nicked (P = 0.021) crab, with an overall significant increase in granulocyte numbers observed in nicked crab when compared to non-nicked (P = 0.023).
J.E. Welsh et al. / Journal of Invertebrate Pathology 112 (2013) 49–56
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Fig. 4. (A) Necrosis (small arrow) and advanced melanised nodules (large arrow). (B) Swelling, haemocyte aggregate and melanisation within a secondary gill lamella from a nicked crab on day 1 (arrow). (C/D) Melanised nodules (arrow) in connective tissue from a nicked crab on day 4. (E/F) Dense bacterial biofilm found on the secondary gill lamella of a nicked crab on day 3 (arrows).
Fig. 5. Mean (±standard error) number of necrotic areas found in all the sampled tissues for nicked and non-nicked crab on each day. Areas were defined as a necrotic region which was surrounded by healthy cells.
4. Discussion Cumulative mortality rates between nicked and non-nicked crab remained the same until day 5. However, mortality rates for nicked crab increased dramatically on day 7. Uglow et al. (2005) reported high mortality rates during post-harvest processes to be due to a combination of factors, which would normally be innocuous on their own. However, some patterns did emerge from their
study. Greater mortalities were recorded 72 h post transportation and during the summer (Wyman et al., 2005; Uglow et al., 2005). In this study an increase in mortality rates after 72 h was not observed, though an increase in mortality was recorded after 168 h (7 days after landing, 6 days after transportation). Uglow et al. (2005) did, however, suggest that differences in crab population condition affected mortalities. A similar investigation incorporating crab landed from the South west and Eastern Irish fisheries would be required to identify any differences in condition of the populations. Temperature on day 7 peaked at 14.9 °C. At peak, the water temperature was elevated for the time of year but below the temperatures that brown crab may experience during summer. Temperature changes can alter metabolic rate and oxygen consumption in brown crab (Frederich and Pörtner, 2000; Uglow et al., 2005), with an increase in one resulting in an increase in the other. Frederich and Pörtner (2000) identified optimal temperatures of 8–17 °C for aerobic performance. However, the addition of factors such as stress and starvation alter oxygen and metabolic rate (Depledge, 1985; Lorenzon et al., 2008). Uglow et al. (2005) showed that oxygen uptake in active or stressed brown crab held at 12 °C was ca. 0.02 mL O2 g 1 h 1 greater than in settled crab. Increased metabolic rate and reduced O2 availability is likely to result in the animal respiring anaerobically. Anaerobic respiration causes a buildup of waste products such as lactate in the haemolymph (Wyman et al., 2005; Uglow et al., 2005; Lorenzon et al., 2008). During this investigation there was a recorded decrease in glucose, and lactate from day 0 to day 2 in both nicked and nonnicked crabs. This was probably due to an acclimatisation period
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Fig. 6. Time course of haemolymph glucose, lactate and refractive index in nicked (solid line) and non-nicked (dashed line) crab. Values shown as mean ± standard error.
Table 2 Changes in mean number of granulocytes (±standard error) for nicked and non-nicked crab over the trial period. Granulocyte numbers in nicked crab ranged from 32.90 ± 12.97 to 40.00 ± 10.22, whereas, non-nicked granulocyte numbers ranged from 31.90 ± 8.21 to 39.60 ± 6.65. Day
0 1 2 3 4 5 7
Granulocytes Nicked
Non-nicked
32.90 + 12.97 40.00 ± 10.22 36.30 ± 5.42 37.60 ± 7.60 33.50 + 6.38 39.40 ± 5.60 37.30 ± 10.06
32.10 + 5.65 37.00 ± 10.92 31.90 + 8.21 34.50 ± 6.96 36.30 + 7.72 37.80 ± 6.56 39.60 ± 6.56
in the tanks. However, this does not explain the continual decrease in lactate after day 2 but suggests that the crab were not utilising blood protein reserves for energy, despite the refractive index of haemolymph decreasing during the trial. Nicked crab showed significantly higher (P = 0.022) levels of pathologies than non-nicked crab. Both nicked and non-nicked crab showed an increase in pathologies during the trial. Observations showed that the majority of pathologies were of advanced stages of phagocytosis, with few naïve stages such as haemocyte aggregates. Advanced stages were characterised by localised necrotic tissue with a darkened, melanised core and encirculating, elongated haemocytes (Smith and Ratcliffe, 1980b; Bayne, 1990; Vogan et al., 2001). Melanisation has previously been associated
with phagocytic clearing of bacterial infections in crustaceans (Smith and Ratcliffe, 1980a,b; Ratcliffe and Rowley, 1981; Johnson, 1980, 1987; Johansson et al., 2000; Lee and Söderhäll, 2002; Vogan et al., 2001). This suggests that the majority of pathologies were cause by infections either before capture, or in the early stages of post-harvest processes. However, the few haemocyte aggregates which were observed were likely to be a result of post-harvest activities. As experimental animals were obtained from their natural environment, it is expected that animals would have acquired a certain number of pathologies, which were identified at time point zero. Both non-nicked and nicked animals did demonstrate an increase in the number of pathologies over time, apart from day 4, where a significant increase in the number of pathologies was observed in nicked crab. It would appear that a stress was experienced by both groups in around day 4, as indicated by the glucose levels; however the nicked animals acquired more pathologies, whilst the non-nicked remained stable. This would suggest that the experimental group became compromised due to nicking and are therefore unable to deal with additional challenges when compared to the non-nicked. In addition, an increase in mortality was observed from day 4 onwards (Fig. 1); however mortalities were not subjected to histological examination. Prevalence of muscle necrosis varied with the worst cases observed in nicked animals. Necrosis has previously been reported in the muscle of Nephrops norvegicus (Stentiford and Neil, 2000). Muscle necrosis has also been associated with Enterococcus like bacterium (Cheng and Chen, 1998) and post capture damage and stressors (Stentiford and Neil, 2000). Furthermore, during this investigation infiltration of haemocytes were observed within claw muscle bundles of nicked crab. This finding is consistent with necrosis descriptions by Stentiford and Neil (2000) and Cheng and Chen (1998). More obvious damage showed that blackening and scaring of claw tissue occurred within the first 7 days of the trial. This finding showed that extreme melanisation occurred quicker than in the experiments by Jacklin and Combes (2007), who noted that the claw meat from nicked crab started to blacken after 10 days in storage. The implications of damage to the claw muscle caused by nicking are twofold. Firstly, the damage results in a reduction in the animal’s health and possibly reduced survivability. Such mechanical damage to the claw muscle initiates the host’s innate immune response (Metchnikoff, 1901; Bang, 1970; McKay and Jenkin, 1970; Smith and Ratcliffe, 1980a,b: Tauber, 2003) and when coupled with stressors, such as storage and handling, the host ability to elicit a further innate immune response to pathological challenges becomes compromised. This has been shown in mammals (Maslanika et al., 2009), invertebrates (Mydlarz et al., 2008) and crustaceans (Stewart and Zwicker, 1972; Le Moullac and Haffner, 2000; de la Vega et al., 2007). Secondly, the claw muscle is the most valuable product of brown crab. Discoloration and a reduction in quality of claw muscle results in a lower valued or unmarketable product. Non-invasive methods of claw retention would alleviate damage caused to the internal claw tissue. Previous studies have successfully used banding (Haefner, 1971) and wire (Newman and Ward, 1973) to retain crab claws. However, neither method has been shown to successfully work on brown crab due to claw morphometrics. There was an observed change in hepatopancreatic tubule lumen size during the trial. The increase appeared to be due to a reduction in the size of Restzellen and Blasenzellen cells (R-cells and B-cells respectively) (Johnson, 1980). R and B cells store lipids and glycogen, therefore, it is expected that there would be a reduction in size due to starvation whilst the animals are in the commercial ponds (Papathanassiou and King, 1984; Nishida et al., 1995; Uglow et al., 2005).
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Encirculating granulocyte numbers in non-nicked crab were comparable to levels in non-nicked crab (32.90 ± 12.97 and 32.10 ± 5.65 respectively) at time point 0. On day 1, granulocyte numbers increased for both groups. However, only nicked crab showed a significant increase in granulocyte numbers when compared to non-nicked (P = 0.023). Hose et al., 1990 showed that hyaline cells are responsible for lysis and coagulation, while granulocytes initiate phagocytosis of foreign material (Hose and Martin, 1989) using prophenoloxidase (Cerenius and Söderhäll, 2004). Thus, granulocyte numbers are a potential measurement of possible infection levels. Observations made by Jussila et al. (2001) showed that increased levels of semi-granulocytes (counted as granulocytes in this study) decreased clotting time of haemolymph. Reduced clotting times has also been associated with stress (Jussila et al., 2001). This suggests that nicked crab, which showed higher numbers of encirculating granulocytes, may have been undergoing a continuous pathogen challenge. However, contrary to Jussila et al. (2001), other studies identified a decrease in circulating haemocyte numbers during an infection (Smith and Söderhäll, 1983; Lorenzon et al., 2000). This may have been due to circulating hyaline cells being removed for coagulation, resulting in higher levels of granulocytes in total haemocyte counts. Nonetheless, our assumptions need to be validated. Furthermore, results from Paterson et al. (2005) identified an increase in granulocytes with a decrease in lactate. This study however, did not show a correlation between granulocyte numbers and lactate levels. The industrial practice of nicking in the Irish crab fishery has been shown to have a negative effect on the animal’s physiology and host defence mechanism, resulting in elevated levels of pathologies, glucose and circulating granulocytes. Continued use of claw restraints is recommended in order to prevent inter-species damage during post-harvest storage. Also, further research and development is required to source an alternative, non-invasive method to nicking as a means of claw retention. Acknowledgments The study was funded by Enterprise Ireland, ARE ShellTec Research Centre and the Galway-Mayo Institute of Technology. Thanks to Pat Freeney and Brendan Allen for their provision and assistance with the ponds, and the anonymous reviewers for their valued input. References Anon, 2009. Shellfish stocks and fisheries: Review 2009. Marine Institute, Rinville, Oranmore, Galway, 84pp. ISBN 978-1-902895-42-0. Bang, F.B., 1970. Disease mechanisms in crustacean and marine arthropods. Am. Fish. Soc. 5, 383–404 (Special Publication). Barrento, S., Marques, A., Pedre, S., Vaz-Pires, P., Nunes, M.L., 2008. The trade of live crustaceans in Portugal: space for technological improvements. ICES J. Mar. Sci. 65, 551–559. Barrento, S., Marques, A., Vaz-Pires, P., Nunes, M.L., 2010. Live shipment of immersed crabs Cancer pagurus from England to Portugal and recovery in stocking tanks: stress parameter characterization. ICES J. Mar. Sci. 67, 435– 443. Battistella, S., Bonivento, P., Amirante, G.A., 1996. Hemocytes and immunological reactions in crustaceans. Ital. J. Zool. 63, 337–343. Bayne, C.J., 1990. Phagocytosis and non-self recognition in Invertebrates. BioScience 40, 723–731 (10. Comparative Immunology). Beaven, G.E., Truitt, R.V., 1940. Crab mortality on Chesapeake Bay shedding floats. Chesapeake BioI. Lab. Contrib. 33, 14. Bergmann, M., Taylor, A.C., Moore, P.G., 2001. Physiological stress in decapod crustaceans (Munida rugosa and Liocarcinus depurator) discarded in the Clyde Nephrops fishery. J. Exp. Mar. Biol. Ecol. 259, 215–229. Cerenius, L., Söderhäll, K., 2004. The prophenoloxidase-activating system in invertebrates. Immunol.Rev. 198 (1), 116–126, 11. Cheng, W., Chen, J.C., 1998. Isolation and characterization of Enterococcus-like bacterium causing muscle necrosis and mortality with Macrobrachium rosenbergii in Taiwan. Dis. Aquat. Org. 34, 93–101.
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Pathological and physiological effects of nicking on brown crab (Cancer pagurus) in the Irish crustacean fishery Jennifer E. Welsh, Pauline A. King, Eugene MacCarthy ⇑ Galway-Mayo Institute of Technology, Galway, Ireland
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Article history: Received 2 January 2012 Accepted 15 August 2012 Available online 24 August 2012 Keywords: Brown crab (Cancer pagurus) Nicking Necrosis Pathology Handling Storage
a b s t r a c t Nicking is used in fisheries to immobilize claws of brown crab (Cancer pagurus) in order to prevent cannibalism and fighting during storage. Nicking fractures the apodemes creating an open wound and damage to the internal claw tissues, which is the most valuable product of brown crab. In turn, this results in a reduction of quality of product and possibly compromises the host’s defence mechanisms to other physiological challenges experienced throughout the post-harvest process. This study assessed the effects of nicking on the physiology and pathology of brown crab from the Irish fishery over 7 days. Results showed significantly elevated levels of muscle necrosis (P = 0.005), total pathologies (P = 0.022) and encirculating granulocytes in nicked crab compared to non-nicked crab. Mean glucose (212.0 lg/mL ± 108.4), lactate (36.52 lg/mL ± 38.74) and RI (11.05n ± 1.78) levels were higher in nicked crab indicating increased stress levels. Overall, histology results showed a significantly higher (P = 0.022) occurrence of pathologies, such as melanised nodules, in nicked animals. In addition to an observed reduction in the quality of claw muscle, nicked crab also showed significantly higher (P = 0.005) levels of necrosis in claw muscle. From the results of this study it is recommended that alternative retention methods are used. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Nicking is the typical method of immobilizing claws (chelae) of brown crab (Cancer pagurus) in Ireland. Nicking renders the claw non-functional by fracturing the apodemes (tendon). It is employed by fishers to prevent damage whilst the animals are grouped and for ease of processing (Newman and Ward, 1973; Jacklin and Combes, 2007; Barrento et al., 2008). Two main techniques are used: ‘French’ nicking, cuts the apodemes between the upper and lower dactyl (Haefner, 1971) while ‘English’ nicking, cuts the apodemes between the palm and pincer (Jacklin, 1998). Nicking is not commonly used in the fishery of other crab or lobster species but is typically substituted by banding the claws. The sloped morphology of brown crab claws means that, within the fishery, banding is not practical. Nicking has been shown to create wounds, damage tissue, alter molting and haemolymph clotting processes, and cause swelling (Beaven and Truitt, 1940; Haefner, 1971; Jacklin, 1998; Jacklin and Combes, 2007). Aerial exposure and other post-harvest processes may reduce the haemolymph clotting capacity (Jacklin, 1998; Jacklin and Combes, 2007). Therefore, Jacklin and Combes (2007) suggested that animals should be placed immediately into seawater after nicking to encourage haemolymph coagulation. ⇑ Corresponding author. Present address: Marine and Freshwater Research Centre (MFRC), Galway-Mayo Institute of Technology, Dublin Road, Galway, Ireland. E-mail address: [email protected] (E. MacCarthy). 0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2012.08.006
However, extended coagulation periods may proliferate the time for which bacterial infections can manifest within the claw tissue, increasing the risk of infection by opportunistic pathogens (Jacklin and Combes, 2007). In addition, ‘English’ nicking results in increased bleeding (Jacklin and Combes, 2007). The claw contains the most valuable product of brown crab, therefore, any damage or deterioration caused to the claw muscle results in a lower quality product. Several studies have documented the transport chain from time of capture for live shellfish from capture to consumer (Hearn, 2005; Uglow et al., 2005; Barrento et al., 2008), where total holding and transport times for some crab species vary between 2 and 14 days (Hearn, 2005; Uglow et al., 2005). In general, crabs are nicked on board the fishing vessel at the point of capture and are subjected to a variety of handling techniques and stressors (Paterson and Spanoghe, 1997; Lorenzon et al., 2008). Post capture stressors include aerial exposure, emersion, vibrations, and environmental (temperature and chemical) changes (Spicer et al., 1990; Paterson and Spanoghe, 1997; Taylor et al., 1997; Lorenzon et al., 2008). Damage has, however, been shown to be dependent on the size, age, diet and reproductive state of the individuals (Kruse et al., 1994). Stressors from post-harvest processes may ultimately result in increased prevalence of infections and mortalities. Current industry practices does not require for mortalities to be documented on a daily basis during transportation and storage, however several studies have observed varying degrees of mortality throughout the market chain (Uglow et al., 1986; Stentiford et al., 2002; Stentiford and Sheilds, 2005; Jacklin and Combes,
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2007; Barrento et al., 2008, 2010). Whilst no publications have noted a direct link between nicking and mortalities, it is hypothesised that such wounds inflicted on the crab may contribute to a reduction in overall fitness of the animals. In recent years physiological parameters have been used to assess stress levels in shellfish during the live marketing chain (Söderhäll and Smith, 1986; Taylor et al., 1997; Paterson and Spanoghe, 1997; Fotedar et al., 2006; Gornik et al., 2008; Lorenzon et al., 2008; Barrento et al., 2010). Haemolymph glucose, lactate (used to indicate haemolymph protein concentration) and refractive index have proven to be efficient parameters in measuring crustacean stress levels (Durand et al., 2000; Bergmann et al., 2001; Jussila et al., 2001; Speed et al., 2001; Wyman et al., 2005; Lorenzon et al., 2007). Previously, blood refractive index has been used as a fast response detection method for determining the nutritional condition of decapods (Oliver et al., 2001) due to its correlation with protein concentrations and muscle tissue (Stewart and Li, 1969; Moore et al., 2000). Barrento et al. (2010) noted that monitoring stress responses identifies areas for improvement and may result in the development of enhanced procedures, and therefore, a reduction in mortality rates. Furthermore, quantification of hyaline and granulocyte cells in the form of total or differential haemocyte counts have been used to gauge the health of crustaceans (Smith and Chisholm, 1992; Lorenzon et al., 2000, 2007, 2008). Semi-granulocytes and granulocytes store prophenoloxidase, which initiates pro-PO system, an in vitro phagocytosis reaction (Johansson and Söderhäll, 1985; Söderhäll and Aspan, 1993). During the pro-PO system granulocytes encapsulate foreign material, including bacteria and other pathogens, resulting in melanisation of the tissue (Smith and Söderhäll, 1983; Hose and Martin, 1989; Battistella et al., 1996). After encapsulation both granulocytes and semi-granulocytes initiate melanin synthesis (Smith and Söderhäll, 1983) which breaks down the foreign object. Semi-granulocyte cells respond specifically to polysaccharides found in microbes (Johansson and Söderhäll, 1985). In addition to physiological parameters histological methods can be imperative in assessing tissue degradation. Histology has previously been used to evaluate the effects of post-harvest processes on crustaceans (Paterson et al., 1997; Stentiford and Neil, 2000; Ridgway et al., 2006) and is particularly valuable when assessing muscle necrosis. Necrosis may be a result of damage caused by post capture handling and potentially lead to mortalities further down the marketing chain (Stentiford and Neil, 2000). Finally, brown crab are commercially important throughout Europe and are considered to be the most commercially valuable shellfish species within Ireland (Tully et al., 2006) with landings of ca. 6284 tonnes, worth €13,228,812 in 2008 (Anon, 2009). Little consideration has been previously given to the effects of nicking as a point of entry for opportunistic bacteria and potential pathogens. In keeping with commercial practices, the pathological and physiological effects of nicking on brown crab in the Irish fishery were assessed over a 7 day period. 2. Materials and methods 2.1. Experimental design Two hundred and seventy female brown crab (550–800 g) were caught off Malin Head, Co. Donegal, Ireland. Of these, 135 crabs were nicked using the commercial French method and 135 were banded using 5–10 cm inner tube lengths. Ten nicked crab and 10 non-nicked were sampled on landing (day 0) and the tissue stored for histological analysis (as described in Section 2.2). In addition, haemolymph samples were obtained (as described in Section 2.3) from five non-nicked and five nicked animals for glucose and lactate analysis.
Remaining crabs were transported by road in fish boxes to Clarinbridge, County Galway (ca. 331 km; 5 h) at 6 °C. On arrival both the non-nicked and nicked crab were placed in four 200 L tanks with shared re-circulated natural seawater. Transportation and holding methods utilised in this study were similar to the methods and timescale employed by the Irish crab fishery. During this storage period crab were not fed and the water quality was monitored throughout the trial. Parameters measured included temperature, salinity, pH, Nitrate, Nitrite and Ammonia using Hach Lang DR 800 colorimeter. In addition, mortalities, limb loss and general crab condition were recorded. 2.2. Histology Following anesthetising on ice 10 nicked crab were dissected at 0, 1, 2, 3, 4, 5 and 7 days post nicking, with 10 corresponding controls (non-nicked crab) also dissected per sampling point. Approximately 7 mm2 samples of heart, hepatopancreas, gill, gut, gonad, claw muscle and epidermis were placed into Davidsons seawater fixative (Hopwood, 1996; Howard et al., 2007) for 24 h, after which they were stored in 70% ethanol pending further investigation. Due to the large number of crabs and the variety of methods being used for this study, the realistic number of tissues per crab that could be sampled was limited to seven. However, the sampled tissues were chosen because they provide a good overview of the animal’s health status. Processing was undertaken using standard histological procedures (Feist et al., 2004). Following embedding, 3–5 lm sections were mounted on slides and stained using the Hematoxylin and Eosin Y (H&E) stain method as in Johnson (1980). The tissues were then examined using a light microscope (Olympus BX41) and camera system (Olympus E330 and Olympus Cell-a software). 2.3. Haemolymph D-glucose and L-lactate analysis Haemolymph was extracted (0.2 mL) using a 19 gauge syringe from the basis between the ischium and coax from five nicked and five non-nicked crab on day 0, 2, 4 and 7. Crab subjected to haemolymph extraction were subsequently removed from experimental tanks. Extracted haemolymph was deproteinated by mixing with equal volumes of ice-chilled 0.6 M perchloric acid (Aldrich) and stored at 20 °C until required. Prior to analysis deproteinated samples were defrosted and centrifuged at 8000g for 3 min and the supernatant removed and collected in microtubes. The supernatant of each sample was neutralised by adding 7 lL of potassium bicarbonate (2 M) and gently shaken for 1 min. To complete neutralisation the supernatant was allowed to stand for 5 min at 4 °C. Once neutralisation was complete the precipitated potassium perchlorate formed was separated by centrifugation at 8000g for 1 min and the supernatant collected in microtubes. Quantification of haemolymph D-glucose was carried out by diluting 50 lL of the supernatant with 100 lL of deionised water. 100 lL of the diluted supernatant was transferred to a 96 well plate (Costar). A standard curve was constructed with D-glucose (Sigma) serially diluted (0–80 lg/mL) in deionised water. 200 lL of the assay reagent (0.8 mL of 15.75 mM o-Dianisidine dihydrochloride was added to 39.2 mL of Glucose Oxidase/Peroxidase Reagent (Sigma)) was applied to each well and incubated at 37 °C for 30 min. The reaction was stopped by the addition of 200 lL of 12 N Sulphuric acid and the product absorbance read at 540 nm (Labsystems Multiskan RC). The absorbance values of the standard curve were entered into the program Graph Pad Prisim 4 (GraphPad Software) and the D-glucose concentrations of individual haemolymph samples determined.
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Circulating haemolymph L-lactate levels were measured by taking 2.5 lL of the diluted deproteinated supernatant as outlined above and placed in individual wells of a 96-well plate (Costar). A standard curve was constructed with L-lactate (Trinity Biotech) serially diluted (0–40 lg/mL). 250 lL of L-lactate reagent (Trinity Biotech) was applied to each well and incubated for 5 min at 25 °C. Product absorbance was read at 540 nm (Labsystems Multiskan RC). As outlined for D-glucose, the L-lactate concentrations were determined using the program Graph Pad Prisim 4. 2.3.1. Refractive index Haemolymph was extracted (0.2 mL) using a 19 gauge syringe and placed directly onto the refractometer (E-Line ATC refractometer, Bellingham & Stanley Ltd., UK) and read to assess condition. The refractometer was blanked using deionised water prior to haemolymph analysis. Fig. 1. Percentage cumulative mortality rates over the duration of the experiment (7 days) in both nicked (solid line) and non-nicked (dashed line) crab.
2.3.2. Haemocyte counts Separate samples (0.2 mL) of haemolymph from those taken for the assays were extracted using a 19 gauge syringe and placed directly into 4% neutral buffered formalin (NBF) for fixation. Fixed haemolymph was smeared onto slides and analysed for granulocyte counts using a light microscope (Olympus BX41) and camera system (Olympus E330 and Olympus Cell-a programme). For the purpose of this study, semi-granulocytes were counted as granulocytes. 2.4. Statistics Descriptive statistics using Minitab 15 were carried out on all the data. After testing for normality, parametric and transformed non-parametric data was subjected to an analysis of variance with Tukey’s family error rate. Results that could not be transformed to parametric data was subjected to Wilcoxon signed ranking tests. 3. Results Water quality parameters are shown in Table 1. Temperature ranged from 12.80 °C to 14.90 °C; salinity from 31.00 ppt to 32.50 ppt; pH from 6.23 to 7.10; Nitrate from 5.50.0 mg/L to 24.30 mg/L; Nitrite from 9.00 mg/L to 67.00 mg/L and Ammonia from 0.64 mg/L to 0.73 mg/L. Fig. 1 shows cumulative mortality rates throughout the investigation. Up to day 5, mortality rates were identical in both nicked and non-nicked animals, with an increase in mortality rates observed on day 7 in nicked crab (Fig. 1). Other observations made throughout the duration of the experiment include blackening of the claw tissue around the open wound in nicked crab Fig. 2. This was documented in up to 80% of the nicked crab after day 5, where the extent to which internal tissue is exposed to external factors (Fig. 2C and D). Though not significant, claw loss was greater in nicked crab (mean = 1.43 ± 1.21) compared to non-nicked crab (mean = 0.29 ± 0.49).
3.1. Histology The number of pathologies observed over time in nicked and non-nicked crab is shown in Fig. 3. Both nicked and non-nicked crab showed pathologies throughout the trial. Pathologies observed included granulomas, haemocyte aggregates, necrosis and gill biofilms. The latter are presented in Fig. 4. Pathology data was non-parametric and was therefore subjected to Wilcoxon signed rank tests. Analysis showed a significant difference in pathologies between nicked and non-nicked crab (P = 0.022) over the seven day trial period, with more pathologies observed in nicked crab (collective mean = 15.00 ± 4.24) than non-nicked crab (collective mean = 8.30 ± 2.33) (Fig. 3). In nicked crab the greatest difference was seen between day 0 (mean = 4.50 ± 0.00) and day 4 (mean = 17.30 ± 29.96). However, one animal on day 4 resulted in an outlier with 97 pathologies (Fig. 3). This anomaly showed numerous areas of phagocytises at varying stages of melanisation (from granuloma to melanised nodules) within the connective tissue surrounding the gut. Non-nicked crab recorded the greatest difference in observed pathologies between day 0 (mean = 1.50 ± 0.00) and day 7 (mean = 9.00 ± 1.75). Interestingly, as shown in Fig. 5, non-nicked crab showed a consistent increase in mean number of pathologies unlike nicked crab which showed a more erratic pattern over the 7 days. Necrosis was significantly (P = 0.005) and consistently higher in the claw muscle of nicked crab compared to non-nicked crab (Fig. 5). Over twice the amount of melanisation occurred in the hepatopancreas of nicked crab than in non-nicked crab (mean = 0.19 ± 0.16; mean 0.06 ± 0.06 respectively). In addition, some nicked crab showed haemocyte infiltrations between fibre bundles. Other gross observations showed an increase in hepatopancreatic tubule lumen size and a reduction in R and B cells. Most
Table 1 Water quality parameter measurements taken over the experimental period. Day
Temp. (°C)
Salinity (ppt)
pH
Nitrate (mg/L)
Nitrite (mg/L)
Ammonia (mg/L)
0 1 2 3 4 5 7
12.80 12.80 12.80 12.70 12.70 12.70 14.90
32.50 32.50 32.00 31.30 31.40 31.00 31.10
7.10 7.10 7.06 7.05 7.10 7.01 6.23
550 5.50 20.60 24.30 23.00 24.30 24.30
29.00 29.00 67.00 23.00 47.00 9.00 9.00
0.64 0.64 0.73 0.73 0.73 0.73 0.73
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Fig. 2. (A) The non-nicked tendon between the upper and lower dactyl (arrow) showing no tissue damage (B; arrow); (C) The point where the tendon is fractured (arrow) during French nicking; (D) The blackening of muscle within the claw (arrow). All crabs photographed were from day 7.
Fig. 3. The mean number (±standard error) of pathologies identified in the sampled tissues from nicked (solid line) and non-nicked (dashed line) crabs.
samples showed normal levels of reserve inclusion cells and mature oocytes. 3.2. Haemolymph analysis There was no significant difference between glucose, lactate or RI values in nicked crab compared to non-nicked crab (Fig. 6). However, mean values were higher in nicked crab. Nicked crab showed mean values of 212.0 lg/mL (±108.4), 36.52 lg/mL
(±38.74) and 11.05n (±1.78) for glucose, lactate and RI respectively (Fig. 6). Glucose values for non-nicked crab significantly increased (P = 0.001) over the trial period from 133.60 lg/mL (±39.41) to 303.40 lg/mL (±62.36). Nicked crab glucose values declined from 243.17 lg/mL (±99.37) on day 0 to 151.70 lg/mL (±31.19) on day 7 (Fig. 6). Initial lactate values in nicked and non-nicked crab on day 0 were 89.51 lg/mL (±29.86) and 59.02 lg/mL (±28.56) respectively, decreasing to 6.33.05 lg/mL (±2.42) and 2.50 lg/mL (±0.89) on day 7 (Fig. 6). Post hock tests showed a highly significant difference (P = 0.001) between days 0 and 7 in non-nicked but not in nicked crab. Refractive index (RI) values for nicked crab significantly (P = 0.002) decreased between day 0 (11.46 ± 1.99) and day 7 (9.76 ± 0.96). Non-nicked crab refractive index values did not significantly increase over the trial period, instead mean values fluctuated from 11.62n (±1.25) on day 0 to 10.78n (±0.57) on day 7 (Fig. 6). Changes in encirculating granulocyte numbers were investigated in both nicked and non-nicked crab (Table 2). Granulocytes in non-nicked crab were comparable to levels in non-nicked crab (32.90 ± 12.97 and 32.10 ± 5.65 respectively) at time point 0 (Table 2). An initial increase in granulocyte numbers was observed on day 1 in both groups of crab and this slight increase was maintained through the remaining time points in both nicked (P = 0.032) and non-nicked (P = 0.021) crab, with an overall significant increase in granulocyte numbers observed in nicked crab when compared to non-nicked (P = 0.023).
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Fig. 4. (A) Necrosis (small arrow) and advanced melanised nodules (large arrow). (B) Swelling, haemocyte aggregate and melanisation within a secondary gill lamella from a nicked crab on day 1 (arrow). (C/D) Melanised nodules (arrow) in connective tissue from a nicked crab on day 4. (E/F) Dense bacterial biofilm found on the secondary gill lamella of a nicked crab on day 3 (arrows).
Fig. 5. Mean (±standard error) number of necrotic areas found in all the sampled tissues for nicked and non-nicked crab on each day. Areas were defined as a necrotic region which was surrounded by healthy cells.
4. Discussion Cumulative mortality rates between nicked and non-nicked crab remained the same until day 5. However, mortality rates for nicked crab increased dramatically on day 7. Uglow et al. (2005) reported high mortality rates during post-harvest processes to be due to a combination of factors, which would normally be innocuous on their own. However, some patterns did emerge from their
study. Greater mortalities were recorded 72 h post transportation and during the summer (Wyman et al., 2005; Uglow et al., 2005). In this study an increase in mortality rates after 72 h was not observed, though an increase in mortality was recorded after 168 h (7 days after landing, 6 days after transportation). Uglow et al. (2005) did, however, suggest that differences in crab population condition affected mortalities. A similar investigation incorporating crab landed from the South west and Eastern Irish fisheries would be required to identify any differences in condition of the populations. Temperature on day 7 peaked at 14.9 °C. At peak, the water temperature was elevated for the time of year but below the temperatures that brown crab may experience during summer. Temperature changes can alter metabolic rate and oxygen consumption in brown crab (Frederich and Pörtner, 2000; Uglow et al., 2005), with an increase in one resulting in an increase in the other. Frederich and Pörtner (2000) identified optimal temperatures of 8–17 °C for aerobic performance. However, the addition of factors such as stress and starvation alter oxygen and metabolic rate (Depledge, 1985; Lorenzon et al., 2008). Uglow et al. (2005) showed that oxygen uptake in active or stressed brown crab held at 12 °C was ca. 0.02 mL O2 g 1 h 1 greater than in settled crab. Increased metabolic rate and reduced O2 availability is likely to result in the animal respiring anaerobically. Anaerobic respiration causes a buildup of waste products such as lactate in the haemolymph (Wyman et al., 2005; Uglow et al., 2005; Lorenzon et al., 2008). During this investigation there was a recorded decrease in glucose, and lactate from day 0 to day 2 in both nicked and nonnicked crabs. This was probably due to an acclimatisation period
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J.E. Welsh et al. / Journal of Invertebrate Pathology 112 (2013) 49–56
Fig. 6. Time course of haemolymph glucose, lactate and refractive index in nicked (solid line) and non-nicked (dashed line) crab. Values shown as mean ± standard error.
Table 2 Changes in mean number of granulocytes (±standard error) for nicked and non-nicked crab over the trial period. Granulocyte numbers in nicked crab ranged from 32.90 ± 12.97 to 40.00 ± 10.22, whereas, non-nicked granulocyte numbers ranged from 31.90 ± 8.21 to 39.60 ± 6.65. Day
0 1 2 3 4 5 7
Granulocytes Nicked
Non-nicked
32.90 + 12.97 40.00 ± 10.22 36.30 ± 5.42 37.60 ± 7.60 33.50 + 6.38 39.40 ± 5.60 37.30 ± 10.06
32.10 + 5.65 37.00 ± 10.92 31.90 + 8.21 34.50 ± 6.96 36.30 + 7.72 37.80 ± 6.56 39.60 ± 6.56
in the tanks. However, this does not explain the continual decrease in lactate after day 2 but suggests that the crab were not utilising blood protein reserves for energy, despite the refractive index of haemolymph decreasing during the trial. Nicked crab showed significantly higher (P = 0.022) levels of pathologies than non-nicked crab. Both nicked and non-nicked crab showed an increase in pathologies during the trial. Observations showed that the majority of pathologies were of advanced stages of phagocytosis, with few naïve stages such as haemocyte aggregates. Advanced stages were characterised by localised necrotic tissue with a darkened, melanised core and encirculating, elongated haemocytes (Smith and Ratcliffe, 1980b; Bayne, 1990; Vogan et al., 2001). Melanisation has previously been associated
with phagocytic clearing of bacterial infections in crustaceans (Smith and Ratcliffe, 1980a,b; Ratcliffe and Rowley, 1981; Johnson, 1980, 1987; Johansson et al., 2000; Lee and Söderhäll, 2002; Vogan et al., 2001). This suggests that the majority of pathologies were cause by infections either before capture, or in the early stages of post-harvest processes. However, the few haemocyte aggregates which were observed were likely to be a result of post-harvest activities. As experimental animals were obtained from their natural environment, it is expected that animals would have acquired a certain number of pathologies, which were identified at time point zero. Both non-nicked and nicked animals did demonstrate an increase in the number of pathologies over time, apart from day 4, where a significant increase in the number of pathologies was observed in nicked crab. It would appear that a stress was experienced by both groups in around day 4, as indicated by the glucose levels; however the nicked animals acquired more pathologies, whilst the non-nicked remained stable. This would suggest that the experimental group became compromised due to nicking and are therefore unable to deal with additional challenges when compared to the non-nicked. In addition, an increase in mortality was observed from day 4 onwards (Fig. 1); however mortalities were not subjected to histological examination. Prevalence of muscle necrosis varied with the worst cases observed in nicked animals. Necrosis has previously been reported in the muscle of Nephrops norvegicus (Stentiford and Neil, 2000). Muscle necrosis has also been associated with Enterococcus like bacterium (Cheng and Chen, 1998) and post capture damage and stressors (Stentiford and Neil, 2000). Furthermore, during this investigation infiltration of haemocytes were observed within claw muscle bundles of nicked crab. This finding is consistent with necrosis descriptions by Stentiford and Neil (2000) and Cheng and Chen (1998). More obvious damage showed that blackening and scaring of claw tissue occurred within the first 7 days of the trial. This finding showed that extreme melanisation occurred quicker than in the experiments by Jacklin and Combes (2007), who noted that the claw meat from nicked crab started to blacken after 10 days in storage. The implications of damage to the claw muscle caused by nicking are twofold. Firstly, the damage results in a reduction in the animal’s health and possibly reduced survivability. Such mechanical damage to the claw muscle initiates the host’s innate immune response (Metchnikoff, 1901; Bang, 1970; McKay and Jenkin, 1970; Smith and Ratcliffe, 1980a,b: Tauber, 2003) and when coupled with stressors, such as storage and handling, the host ability to elicit a further innate immune response to pathological challenges becomes compromised. This has been shown in mammals (Maslanika et al., 2009), invertebrates (Mydlarz et al., 2008) and crustaceans (Stewart and Zwicker, 1972; Le Moullac and Haffner, 2000; de la Vega et al., 2007). Secondly, the claw muscle is the most valuable product of brown crab. Discoloration and a reduction in quality of claw muscle results in a lower valued or unmarketable product. Non-invasive methods of claw retention would alleviate damage caused to the internal claw tissue. Previous studies have successfully used banding (Haefner, 1971) and wire (Newman and Ward, 1973) to retain crab claws. However, neither method has been shown to successfully work on brown crab due to claw morphometrics. There was an observed change in hepatopancreatic tubule lumen size during the trial. The increase appeared to be due to a reduction in the size of Restzellen and Blasenzellen cells (R-cells and B-cells respectively) (Johnson, 1980). R and B cells store lipids and glycogen, therefore, it is expected that there would be a reduction in size due to starvation whilst the animals are in the commercial ponds (Papathanassiou and King, 1984; Nishida et al., 1995; Uglow et al., 2005).
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Encirculating granulocyte numbers in non-nicked crab were comparable to levels in non-nicked crab (32.90 ± 12.97 and 32.10 ± 5.65 respectively) at time point 0. On day 1, granulocyte numbers increased for both groups. However, only nicked crab showed a significant increase in granulocyte numbers when compared to non-nicked (P = 0.023). Hose et al., 1990 showed that hyaline cells are responsible for lysis and coagulation, while granulocytes initiate phagocytosis of foreign material (Hose and Martin, 1989) using prophenoloxidase (Cerenius and Söderhäll, 2004). Thus, granulocyte numbers are a potential measurement of possible infection levels. Observations made by Jussila et al. (2001) showed that increased levels of semi-granulocytes (counted as granulocytes in this study) decreased clotting time of haemolymph. Reduced clotting times has also been associated with stress (Jussila et al., 2001). This suggests that nicked crab, which showed higher numbers of encirculating granulocytes, may have been undergoing a continuous pathogen challenge. However, contrary to Jussila et al. (2001), other studies identified a decrease in circulating haemocyte numbers during an infection (Smith and Söderhäll, 1983; Lorenzon et al., 2000). This may have been due to circulating hyaline cells being removed for coagulation, resulting in higher levels of granulocytes in total haemocyte counts. Nonetheless, our assumptions need to be validated. Furthermore, results from Paterson et al. (2005) identified an increase in granulocytes with a decrease in lactate. This study however, did not show a correlation between granulocyte numbers and lactate levels. The industrial practice of nicking in the Irish crab fishery has been shown to have a negative effect on the animal’s physiology and host defence mechanism, resulting in elevated levels of pathologies, glucose and circulating granulocytes. Continued use of claw restraints is recommended in order to prevent inter-species damage during post-harvest storage. Also, further research and development is required to source an alternative, non-invasive method to nicking as a means of claw retention. Acknowledgments The study was funded by Enterprise Ireland, ARE ShellTec Research Centre and the Galway-Mayo Institute of Technology. Thanks to Pat Freeney and Brendan Allen for their provision and assistance with the ponds, and the anonymous reviewers for their valued input. References Anon, 2009. Shellfish stocks and fisheries: Review 2009. Marine Institute, Rinville, Oranmore, Galway, 84pp. ISBN 978-1-902895-42-0. Bang, F.B., 1970. Disease mechanisms in crustacean and marine arthropods. Am. Fish. Soc. 5, 383–404 (Special Publication). Barrento, S., Marques, A., Pedre, S., Vaz-Pires, P., Nunes, M.L., 2008. The trade of live crustaceans in Portugal: space for technological improvements. ICES J. Mar. Sci. 65, 551–559. Barrento, S., Marques, A., Vaz-Pires, P., Nunes, M.L., 2010. Live shipment of immersed crabs Cancer pagurus from England to Portugal and recovery in stocking tanks: stress parameter characterization. ICES J. Mar. Sci. 67, 435– 443. Battistella, S., Bonivento, P., Amirante, G.A., 1996. Hemocytes and immunological reactions in crustaceans. Ital. J. Zool. 63, 337–343. Bayne, C.J., 1990. Phagocytosis and non-self recognition in Invertebrates. BioScience 40, 723–731 (10. Comparative Immunology). Beaven, G.E., Truitt, R.V., 1940. Crab mortality on Chesapeake Bay shedding floats. Chesapeake BioI. Lab. Contrib. 33, 14. Bergmann, M., Taylor, A.C., Moore, P.G., 2001. Physiological stress in decapod crustaceans (Munida rugosa and Liocarcinus depurator) discarded in the Clyde Nephrops fishery. J. Exp. Mar. Biol. Ecol. 259, 215–229. Cerenius, L., Söderhäll, K., 2004. The prophenoloxidase-activating system in invertebrates. Immunol.Rev. 198 (1), 116–126, 11. Cheng, W., Chen, J.C., 1998. Isolation and characterization of Enterococcus-like bacterium causing muscle necrosis and mortality with Macrobrachium rosenbergii in Taiwan. Dis. Aquat. Org. 34, 93–101.
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