Seasonal Variation in the Length-Weight Relationships and Condition Factor of Four Commercially Important Sea Cucumbers Species from Karachi Coast-Northern Arabian Sea

1 The Marine Reference Collection and Resource Centre, University of Karachi, Karachi, Pakistan. 2 Centro Regional de Investigaciones Pesqueras de Yucalpeten, Instituto Nacional de Pesca y Acuacultura, Blvd. del Pescador S/N, Puerto de Abrigo, 97320 Progreso, Yucatan, Mexico. 3 University of Sinop, Fisheries Faculty, Department of Hydrobiology, TR57000 Sinop, Turkey. Abstract Length-weight relationships and condition assessment are a common practice in fisheries analyses; however, such information is unknown for most sea cucumber species. The present study investigated length-weight relationships and relative condition indices of four commercially important sea cucumbers: Holothuria arenicola, Holothuria atra, Holothuria pardalis and Holothuria verrucosa. Additionally, the seasonal variations obtained between three climatic seasons (pre-monsoon, monsoon and post-monsoon) were analyzed. The results showed that the four species grow allometrically, with negative allometric b values ranging from 0.84 to 1.74. There were significant differences of length-weight relationships between climatic seasons for H. arenicola and H. atra. The lower b values were observed during post-monsoon and the higher b values during monsoon in three of the four species. During the post-monsoon, the condition of the bigger individuals of H. arenicola and H. atra was lower than that of the small individuals. The differences in length-weight relationships of H. pardalis and H. verrucosa were not significant between climatic events. The higher temperatures occurred during monsoon and the lower during post-monsoon, but these data only showed a significant correlation with the relative condition factor of H. arenicola. Best condition of the individuals of H. arenicola occurred at temperatures between 22 to 31°C. Studies about environmental variables and characteristics of the substratum type are necessary in order to complement the understanding of the length-weight relationships and condition of these species.


Introduction
Length-weight relationships are commonly used in fisheries biology to convert length measures into weight and to determine the growth characteristics related to those variables.Data analysis on length-weight relationships and condition indices is one of the standard methods more employed in fishery biology and widely discussed in many papers of fish (Le Cren, 1951;Froese, 2006).
Sea cucumbers fisheries are an important source of income for many otherwise resourcelimited communities (Clarke, 2004), principally in Asia.Although worldwide about 66 species of sea cucumbers are harvested (Purcell, 2010), important biological parameters are unknown for most species.One of these parameters is the allometric coefficient, which is obtained from length-weight relationships and can be used to convert length distributions into weights for biomass estimates (Gerritsen & McGrath, 2007).Biomass estimates are utile to assess the status of sea cucumbers stocks (Purcell, 2010).
Total 19 sea cucumber species have been recorded from coastal waters of Pakistan, seven of which are important commercially (Purcell, 2010;Purcell et al., 2013;Purcell et al., 2014).Neither traditional fishery of these echinoderms is exist in Pakistan nor studies on them.Only a few studies are exists (Ahmed & Ali, 2014;Siddique et al., 2014;Haider et al., 2015;Ahmed & Ali 2016;Ahmed et al., 2018).Ahmed et al. (2017) have worked on the heavy metals concentration in Holothurians from the northern Arabian Sea coasts of Pakistan.Thus the biological information on the commercially important species is necessary for management of worldwide sea cucumbers current fisheries.
The goal of our study was to investigate the seasonal variability (pre-monsoon, monsoon, and post-monsoon) of length-weight relationship and the relative condition factor of four commercially important sea cucumbers species: Holothuria arenicola Semper, 1868, Holothuria atra Jaeger, 1833, Holothuria pardalis Selenka, 1867 and Holothuria verrucosa Selenka, 1867 in the coastal waters of Karachi, Pakistan.
For taxonomic studies and identification, morphological features were examined and microscopic studies were conducted.Ossicles were taken from three positions (dorsal and ventral body walls, and tentacles), wet mounts were prepared by placing a small piece of skin tissue on slide and adding few drops of 3.5% bleach, the slides were then rinsed with drops of distilled water.The slides were examined under a microscope at 10x10 magnifications (Nikon LABOPHOT-2).Microphotography was also performed through digital camera (Fujifilm 16 MP).Length (cm) and weight (g) data were collected for each sea cucumber after allowing the sea cucumber to relax in water for 5 min.Total length from mouth to anus was measured to the flexible ruler.Wet weight was measured to the nearest 0.01 g immediately after removing the animal from the water to avoid evisceration.

Data analysis
We compared size frequency distributions between Holothurians from Sunehri and Buleji using the Kolmogorov-Smirnov non-parametric test (Sokal and Rohlf, 1997), discriminated by species.
Length-weight relationships were computed for specimens using general regression method (Ricker, 1973).Growth curves for population were also calculated by the same method used by Ricker (1975).In order to describe relative changes in sea cucumbers morphology we used the allometric equation y = ax b , where a and b are parameters, x is the body length (L) and y the wet weight (W).Parameters were calculated by species with non-linear regression analysis, which is a good tool in allometric studies (Ebert and Russel, 1994).The loss function was evaluated with Marquardt's compromise algorithm (Marquardt, 1963), which minimizes the sum of squares (SSQ) of the difference between sample weights and pseudo-weights (Gayanilo and Pauly, 1997).
Convergence was reached at 50 iterations in each regression.Student's t-test was conducted to test the hypothesis of isometry (b = 3).Allometric growth occurred when either b> 3 (negative) or b< 3 (positive).
We analyzed sea cucumbers condition by the relative condition factor K rel (Le Cren, 1951) as follows: Where W is the weight of an individual sea cucumber and W' is the predicted mean weight for the same sea cucumber as calculated from a composite of length-weight regression throughout the range of the species: Seasonal variations in the length-weight relationships were evaluated through one-way analysis of covariance (ANCOVA), using season (pre-monsoon, monsoon and post-monsoon) as the main factor log 10 weight as the dependent variable and log 10 length as the co-variable.Separate analyses were done by specie.The assumptions of homogeneity of the slopes (parallelism test) and linear relationship between the dependent variable and the covariate were tested.When the parallelism assumption was not met, a separate slope model was used as an alternative.When significant differences were found, multiple comparisons were performed using Tukey tests (HSD) (Zar, 1984;Sokal and Rohlf, 1997).In order to detect seasonal variation (pre-monsoon, monsoon and post-monsoon) in the b and K rel values, we focused on variation in condition with the help of a log K rel vs log L plot (Froese, 2006).All statistical analyses were considered significant at p<0.05 and highly significant at p<0.001.

Results
A total of 302 sea cucumbers were identified, measured and weighted, 53 H. arenicola, 30 H. atra, 157 H. pardalis and 62 H. verrucosa.The total lengths ranged from 12.0 to 48.0 cm (mean 22.6 ± 6.9 cm) for H. arenicola, from 12.0 to 34.0 cm (mean 19.1 ± 5.6 cm) for H. atra, from 4.0 to 16.0 cm (mean 8.7 ± 3.1 cm) for H. pardalis and from 5.0 to 19.0 cm (mean 11.6 ± 3.2 cm) for H. verrucosa.Weights ranged from 16.0 to 136.0 g (mean 59.2 ± 29.6 g), 15.0 to 104.0 g (mean 38.6 ± 21.2 g), 5.0 to 27 g (mean 12.9 ± 4.3 g) and from 8.0 to 32.0 g (mean 16.1 ± 3.9 g), for H. arenicola, H. atra, H. pardalis and H. verrucosa, respectively.The size frequency distributions of the four species in Sunehriare were not different from those of Buleji (Kolmogorov-Smirnov test; Table 1).The Students t-tests showed that the four species grow allometrically (b ≠ 3, p<0.05), with negative allometric b values ranging from 0.84 to 1.74 (Table 2).No length-weight relationship showed b values near three (isometric value).In all cases, the relationship between length and weight was highly significant (p<0.001; Figure 2).For the four species, the lower b values occurred during post-monsoon and the higher b values during monsoon (Table 3).There were significant differences in the length-weight relationship of H. arenicola between pre-monsoon, monsoon and post-monsoon (Separate slopes model, F 2,47 = 12.18, p<0.001).Body weight was significantly higher during post-monsoon than during pre-monsoon and monsoon, when Log 10 L was used as a covariate (Tukey test, p< 0.001) (Figure 3a).According to the log K rel vs log L plot, during post-monsoon, large individuals had a lower condition than small individuals; but during pre-monsoon and monsoon, they had a higher condition (Figure 3b).The climatic event also had a significant effect on the length-weight relationship of H. atra (one-way ANCOVA, F 2,26 = 7.32, p<0.05; Figure 4a), and again body weight was significantly higher during post-monsoon than during pre-monsoon and monsoon, when Log 10 L was used as a covariate (Tukey test, p<0.001).The log K rel vs log L plot showed that large individuals had a higher condition than small individuals during the pre-monsoon but lower during monsoon and post-monsoon, however, these tendencies only were statistically significant during the postmonsoon (Figure 4b).Analysis of covariance found no significant difference in the length-weight relationships between climatic events for H. pardalis (F 2,153 = 3.00, p= 0.05) and H. verrucosa (F 2,58 = 0.44, p= 0.64).The log K rel vs log L plot showed large specimens of H. pardalis had the higher condition than small specimens.In the case of H. verrucosa, small specimens had higher condition than large specimens (Figure 5).The water temperature in the study area was higher during monsoon than during premonsoon and post-monsoon (Figure 6a).Only in H. arenicola, a significant negative correlation (r = 0.34, p< 0.05; Figure 6b) between temperature and K rel was found.

Discussion
In our study, larger specimens of H. arenicola were registered than in the Mediterranean Sea on the Egyptian coast (length range: 5.0-21.0cm; Abdel Razek et al., 2010), even longer than in Karachi, Pakistan in 2011 and2012 (length range: 2.7-41.6 cm;Siddique et al., 2014).The presence of smaller specimens of H. arenicola in Egyptian waters might be due to the over exploitation done by fishing (Abdel Razek et al., 2007); occur the same with Holothuria nobilis (Selenka, 1867) on the Great Barrier Reef, where the average weight of specimens significantly reduces on fished reefs compared to reefs closed to fishing (Uthicke & Benzie, 2000).
The mean size of H. atra adult specimens was higher than mean size found by other authors for this same species (Conand, 1989: 13.3 cm;Ebert, 1978: 13.0 cm;Edwards, 1908: 16.37 cm); however, its mean weight was lower than that found by these authors.The lower weights may be due to that the fact the specimens from coastal waters of Pakistan in this study correspond to the small form of this species (Lee et al., 2014;Chao, 1993).According to Chao et al. (1994), the small form of H. atrais abundant in intertidal and shallow water habitats.In addition, it is possible that the specimens collected in the two rocky shores Sunehri and Buleji coast might be reproducing asexually (Chao, 1993), but there is no data available on this fact in Pakistan.
Mean length of H. pardalis was lower than the average body length reported of adult specimens for this species (roughly 12 to 25 cm; Purcell et al., 2012).Furthermore, 57% adult and 43% juvenile of H. pardalis were collected in our study, indicating that the habitat types of the Sunehri and Bulejicoasts (rocky shores) are conducive for both.In India, the habitat of adult individuals of this same species is different from that of the juveniles (Gadhavi et al., 2014).
H. verrucosa was recently recorded for the first time from the coastal areas of Pakistan (Ahmed et al., 2016), its mean size and weight were 11.6 cm and 16.1 g, respectively, but no information on sizes is currently available for comparison with our results.
In the four species, growth was negative allometric (b< 3) this shows that large specimens changed their body shape to become more elongated (Froese, 2006).Namely, length increment was faster than weight increment.This type of growth occurs in many species of sea cucumbers (Table 4), and H. pardalis and H. verrucosa were for the first time recorded.
Because deposit-feeding sea cucumbers ingest sediments high in organic matter in the natural environment (Slater & Carton, 2009), the lower b values that occurred during postmonsoon in the four species of this study, likely to be due to an effect the climatic event on the food availability and quality of sediment.It has been shown that the grain size in marine sediments of Malaysia, can be higher during post-monsoon than pre-monsoon and that this can affect the content of organic matter in the sediment (Kamaruzzaman et al., 2010).
Therefore, an indicator of fatness or general "well-being" is utile in this case, such as the relative condition factor (K rel ), which indicated that during the post-monsoon, the condition of the bigger individuals of H. arenicola and H. atra was lower than that of the smaller individuals.These findings indicate a possible diminishing of the content of organic matter in the sediment during the post-monsoon, which can be affecting the "well-being" of the bigger sea cucumbers.
In the particular case of H. arenicola in Buleji, the higher gonad index values occur during summer (monsoon) and the lower during winter (post-monsoon) (Siddique et al., 2014), coinciding in this study with the higher K rel values and lower K rel values, respectively.These results suggest that the reproduction of H. arenicola could be in synchrony with the abundance of food.
The temperature also plays an important role in the reproductive events and the abundance of food (Morgan, 2000), but only data of H. arenicola showed a significant negative correlation with the relative condition factor K rel .The higher temperatures occurred during monsoon and the lower during post-monsoon, but the better condition of the individuals of H. arenicola occurred at temperatures between 22 to 31°C.The differences of the length-weight relationships of H. pardalis and H. verrucosa were not significant between climatic events, but large specimens of H. pardalis had a better condition than that of the small specimens and vice-versa in specimens of H. verrucosa.Therefore, although both small and large sea cucumbers share the same habitat in the coastal waters of Karachi, Pakistan, size distribution before collecting them could be different perhaps by the characteristics of substratum type.This type of studies has been conducted for Holothuria scabra in Solomon Islands, where it was shown that size distribution was a function of substratum type and depth (Mercier et al., 2000).

Conclusion
The commercially important sea cucumbers found in Coastal waters of Pakistan, grow allometric, form negative, namely, length increment is faster than weight increment.Variations of the parameter b and the relative condition factor K rel that occur between climatic pre-monsoon, monsoon and post-monsoon seasons, indicate changes in the environmental variables of the habitat, which also could have relation with the life history of species, such as the reproductive period and the content of organic matter in the sediments.
Studies about environmental variables and characteristics of the substratum type, as well as the relationships of these with sizes distribution of the sea cucumbers are necessary for understanding the biology and ecology of the species distributed from Karachi coast-Northern Arabian Sea.

Figure 1 .
Figure 1.Study area Map.The red squares indicate the localities where the sea cucumbers H. arenicola, H. atra, H. pardalis and H. verrucosa were collected.

Figure 2 .
Figure 2. Length-weight relationships of four sea cucumbers species with commercial value collected in Karachi, Pakistan.W: weight; L: length; r 2 : determination coefficient.

Figure 5 .
Figure 5. Log-log plot of condition vs length calculated from the weight-length relationships of data taken during collection period in Karachi, Pakistan.

Figure 6
Figure 6.a) Mean temperature taken during pre-monsoon, monsoon and post-monsoon in Karachi, Pakistan; b) Log-log plot of condition vs length calculated from the weight-length relationships of data taken during collection period in Karachi, Pakistan.

Table 1 .
Comparisons between size-frequency distributions and median sizes for Sunahri and Buleji sea cucumbers from Karachi, Pakistan.df = degrees of freedom; Kolmogorov-Smirnov test: when D max > D m,n,α reject the null hypothesis (Ho: No difference between datasets).

Table 2 .
Length-weight relationship parameters for four species of sea cucumbers collected in Karachi, Pakistan.SE: standard error; CL: confidence limit; r 2 : determination coefficient; r: correlation coefficient; a: scaling constant; b: allometric growth coefficient; *p< 0.001.

Table 3 .
Length-weight relationships discriminated by climatic event for the four species of sea cucumbers collected in Karachi, Pakistan.SE: standard error; CL: confidence limit; r 2 : determination coefficient; a: scaling constant; b: allometric growth coefficient; *p< 0.001.

Table 4 .
Parameters from the length-weight relationships recorded for different sea cucumbers species.