Application of a non-invasive technique for estimating clutch size in the Caribbean spiny lobster Panulirus argus (Latreille, 1804)

Application of a non-invasive technique for estimating clutch size in the Caribbean spiny lobster... Abstract We investigated the use of a non-invasive method of fecundity estimation for the Caribbean spiny lobster, Panulirus argus (Latreille, 1804), using a method previously described for the American clawed lobster, Homarus americanus (Milne Edwards, 1837). This method only requires the removal of a small subsample of eggs from each individual as compared to traditional gravimetric methods that strip females of their entire clutch. Comparisons of the non-invasive method with traditional estimates resulted in egg count estimates that differed by a mean of 0.003% but a mean absolute value of 42%. Our results demonstrate the utility of this rapid, inexpensive, and non-destructive method for estimating clutch size for a highly fecund species. Differences between estimates produced using this new method and the traditional gravimetric method are substantial, and therefore it’s accuracy in relation to the traditional gravimetric method remains unknown. Fecundity is a key parameter in population ecology, conservation biology, and the setting of biological references points in fisheries stock assessment (FAO, 1974; Gotelli, 2008). Estimates of fecundity, particularly in marine fishes and invertebrates, has traditionally employed invasive and oft-times destructive methods. Gravimetric estimation methods are most common and involve removing and drying egg masses, counting the number of eggs in a weighed subsample, and dividing the total weight of the egg mass by the mean weight of a single egg (Diaz et al., 1983; Chubb, 2008). Methods that destroy the entire clutch or that require the death of the female, however, preclude the potential for further study of egg and larval development and runs counter to conservation or management measures that aim to maintain spawning stock or promote reproduction. Non-invasive techniques to estimate fecundity using sonography, endoscopy, and photography with image analysis for externally extruded clutches have been attempted in a number of species (Bryan et al., 2007) but these methods can be cost prohibitive, require specialized equipment, or are time consuming. We investigated the use of a non-invasive method to estimate clutch size in the Caribbean spiny lobster, Panulirus argus (Latreille,1804), using the method devised by Currie et al. (2010) for the American lobster, Homarus americanus (Milne Edwards, 1837). The Caribbean spiny lobster is an iconic and economically valuable species; it sustains the primary fishery for 24 Caribbean nations and employs an estimated 50,000 fishers and an additional 200,000 in fishery -related jobs (CRFM, 2011). As a consequence of their high value and market demand, however, many populations of P. argus are currently fully capitalized or overfished (Ehrhardt et al., 2010). Size-fecundity relationships of P. argus have been well studied and there are estimates for populations throughout its range including Brazil (Nascimento & Araújo, 1984), Cuba (Cruz et al., 1987), Florida (Cox & Bertelsen, 1997), and Mexico (Fonseca-Larios & Briones-Fourzán, 1998). Female P. argus larger than 100 mm carapace length (CL) are highly fecund, carrying an estimated hundreds of thousands of eggs in a single clutch (Cox & Bertelsen, 1997). Methods that rely on counting every egg in a clutch are thus impractical for P. argus and other highly fecund species, thus most investigators rely on gravimetric estimations. These methods are still invasive and require not only the removal of the clutch from the female, but also the pleopodal setae to which the eggs are attached. For species like P. argus that produce multiple clutches in a single reproductive season, doing so prohibits the attachment of eggs in subsequent clutches until the female molts again into a reproductive condition. Egg removal thus dooms the reproductive success of this individual for months to years, depending on the reproductive and molting dynamics of the species. Moreover, we are unaware of any study using a gravimetric-based fecundity estimate for lobsters that also included counts of all the eggs in a clutch. The accuracy of this commonly used approach is thus unknown for these highly fecund species. In comparison, the non-invasive method used by Currie et al. (2010) offers a means of estimating clutch size quickly and without specialized equipment, and without the need to remove the entire clutch from an individual, thus allowing the remaining eggs to continue to develop and the females to continue normal reproductive activities. Twenty-two ovigerous females ranging in size from 65.4 to 90.2 mm CL were collected by hand by divers in the Florida Keys, Florida, USA in July 2016 and 2017. Only females with eggs that were bright orange in color (i.e., within the first 1.5 weeks of spawning) were selected. The arrangement of the endopodites (the inner portion of the pleopods) in female P. argus, splits the egg mass into segments (Fig. 1). Larger females typically have four segments, whereas smaller females have three. The length of the entire egg mass and the height of each egg segment was measured using a ruler (Fig. 1). For each egg segment, the ruler was inserted into the center of the egg mass between each segment until it touched the abdomen surface. Figure 1. View largeDownload slide Measurements taken to estimate fecundity: length and height of segments. This figure is available in color at Journal of Crustacean Biology online. Figure 1. View largeDownload slide Measurements taken to estimate fecundity: length and height of segments. This figure is available in color at Journal of Crustacean Biology online. The volume of the entire egg mass was then calculated using the formula for the volume of a cylinder:  volume of egg mass=(πH2L2)×0.4225 (1) where H is the average height of the egg segments and L is the length of the entire egg mass. The volume × length equation is divided by 2 to account for the fact that only half the cylinder contains eggs (i.e., eggs are carried externally). Volume is then multiplied by 0.4225, which is the calculated egg packing density (see below). To calculate mean egg volume, at least 10 eggs were removed from each female and stored in 20 ml scintillation vials containing a 5% formalin-seawater solution. The longest and shortest axes of 10 eggs were then measured under a compound microscope (40× magnification) and averaged to provide an average egg radius. Mean egg volume was thus calculated as:  egg volume=43(πr3) (2) where r is the egg radius. Clutch size was then calculated by dividing the volume of the entire egg mass (equation 1) by mean egg volume (equation 2). To validate estimates of egg counts using the non-invasive method, the entire clutch was removed from the 22 females and preserved in a 5% formalin-seawater solution for 24 h. Eggs were rinsed in freshwater and dried at 60 °C for 48 h. Dried eggs were gently sifted through a 300 μm mesh sieve to remove the funiculae, the connective tissue that attaches eggs to the setae. The eggs were then weighed and five weighed subsamples (of greater than 30 eggs sample–1) were counted under a microscope. Clutch size was calculated as total clutch weight divided by mean average egg weight. A paired t-test was used to compare non-invasive and invasive techniques, with a null hypothesis of no difference in calculated clutch size (R version 3.3.1). The non-invasive method was applied to lobsters caught in the Florida Keys during the summer of 2015 and 2016. The length of egg masses and heights of segment were measured in 102 females (63 to 141 mm CL) with bright orange eggs. The mean egg volume from the 22 sampled lobsters was used in the fecundity estimates, and calculations for clutch size were plotted against female CL. As the relationship between CL and fecundity is typically non-linear, particularly as females get larger, we plotted clutch size relative to female CL (in mm) following log transformation, to obtain the following equation (R2 = 0.7805; standard error = intercept 0.304; slope 0.1520) (Fig. 2).  Log clutch size = 2.8669 log CL–0.3442 (3) Figure 2. View largeDownload slide Estimated mean clutch size (non-invasive method) and carapace length (log transformed) for 102 female Panulirus argus (63–141 mm carapace length) sampled from the Florida Keys, FL. Figure 2. View largeDownload slide Estimated mean clutch size (non-invasive method) and carapace length (log transformed) for 102 female Panulirus argus (63–141 mm carapace length) sampled from the Florida Keys, FL. Back-transformed estimates of clutch size were then compared with estimates from equations for P. argus in the Caribbean: Brazil (Nascimento & Araújo, 1984), Cuba (Cruz et al., 1987), Florida (Cox & Bertelsen, 1997), and Mexico (Fonseca-Larios & Briones-Fourzán, 1998). A reduction in the egg mass volume of 42.25% was incorporated into the egg mass equation to account for egg packing density and to reduce the difference in estimates between the traditional and non-invasive methods. This value was calculated by reducing the density of the egg mass volume by 1% until the smallest percent difference between the traditional method and non-invasive method was found. With this correction, clutch size estimates using the non-invasive method were, on average, only 0.003% lower than gravimetric estimates with a standard error of 13.93% (Table 1). The average difference was nevertheless 42% with a standard error of 10.50% (Table 1) when calculated with absolute values. The mean differences in clutch size estimates between our method and gravimetric-based estimates, however, did not differ significantly (paired t-test, t = 1.3655, df = 21, P = 0.1865). As compared to previously published equations from other locations in the Caribbean, our regression equation produced estimates of clutch size that were markedly lower, a mean percent difference of 62% from the next closest values from Brazil (Fig. 3). Table 1. Fecundity estimates of Panulirus argus using non-invasive and traditional methods. Lobster ID  Lobster carapace length (mm)  Traditional method  Non-invasive method  % Difference  1  84.2  224 712  123 147  45.19  2  82.7  140 329  165 267  –17.77  3  79.5  146 033  218 651  –49.73  4  83.8  192 651  170 754  11.37  5  72.9  90 305  213 144  –136.02  6  65.4  16 498  52 981  –221.14  7  77.6  195 021  206 992  –6.14  8  73  148 434  89 035  40.02  9  83.2  233 377  250 854  –7.49  10  90.2  397 283  489 089  –23.11  11  65.7  181 894  75 559  58.46  12  70.8  142 782  86 453  39.45  13  70.7  88 462  81 357  8.03  14  62  84 650  64 462  23.85  15  56.2  48 940  40 417  17.42  16  75.5  139 292  101 958  26.80  17  80.4  197 810  189 722  4.09  18  66.7  133 594  99 469  25.54  19  78.3  237 700  136 442  42.60  20  69  246 148  96 072  60.97  21  70.1  168 431  147 994  12.13  22  75.1  158 885  86 504  45.56  Mean Standard Error        0.003 13.93  Absolute Mean        41.95  Standard Error        10.50  Lobster ID  Lobster carapace length (mm)  Traditional method  Non-invasive method  % Difference  1  84.2  224 712  123 147  45.19  2  82.7  140 329  165 267  –17.77  3  79.5  146 033  218 651  –49.73  4  83.8  192 651  170 754  11.37  5  72.9  90 305  213 144  –136.02  6  65.4  16 498  52 981  –221.14  7  77.6  195 021  206 992  –6.14  8  73  148 434  89 035  40.02  9  83.2  233 377  250 854  –7.49  10  90.2  397 283  489 089  –23.11  11  65.7  181 894  75 559  58.46  12  70.8  142 782  86 453  39.45  13  70.7  88 462  81 357  8.03  14  62  84 650  64 462  23.85  15  56.2  48 940  40 417  17.42  16  75.5  139 292  101 958  26.80  17  80.4  197 810  189 722  4.09  18  66.7  133 594  99 469  25.54  19  78.3  237 700  136 442  42.60  20  69  246 148  96 072  60.97  21  70.1  168 431  147 994  12.13  22  75.1  158 885  86 504  45.56  Mean Standard Error        0.003 13.93  Absolute Mean        41.95  Standard Error        10.50  View Large Table 1. Fecundity estimates of Panulirus argus using non-invasive and traditional methods. Lobster ID  Lobster carapace length (mm)  Traditional method  Non-invasive method  % Difference  1  84.2  224 712  123 147  45.19  2  82.7  140 329  165 267  –17.77  3  79.5  146 033  218 651  –49.73  4  83.8  192 651  170 754  11.37  5  72.9  90 305  213 144  –136.02  6  65.4  16 498  52 981  –221.14  7  77.6  195 021  206 992  –6.14  8  73  148 434  89 035  40.02  9  83.2  233 377  250 854  –7.49  10  90.2  397 283  489 089  –23.11  11  65.7  181 894  75 559  58.46  12  70.8  142 782  86 453  39.45  13  70.7  88 462  81 357  8.03  14  62  84 650  64 462  23.85  15  56.2  48 940  40 417  17.42  16  75.5  139 292  101 958  26.80  17  80.4  197 810  189 722  4.09  18  66.7  133 594  99 469  25.54  19  78.3  237 700  136 442  42.60  20  69  246 148  96 072  60.97  21  70.1  168 431  147 994  12.13  22  75.1  158 885  86 504  45.56  Mean Standard Error        0.003 13.93  Absolute Mean        41.95  Standard Error        10.50  Lobster ID  Lobster carapace length (mm)  Traditional method  Non-invasive method  % Difference  1  84.2  224 712  123 147  45.19  2  82.7  140 329  165 267  –17.77  3  79.5  146 033  218 651  –49.73  4  83.8  192 651  170 754  11.37  5  72.9  90 305  213 144  –136.02  6  65.4  16 498  52 981  –221.14  7  77.6  195 021  206 992  –6.14  8  73  148 434  89 035  40.02  9  83.2  233 377  250 854  –7.49  10  90.2  397 283  489 089  –23.11  11  65.7  181 894  75 559  58.46  12  70.8  142 782  86 453  39.45  13  70.7  88 462  81 357  8.03  14  62  84 650  64 462  23.85  15  56.2  48 940  40 417  17.42  16  75.5  139 292  101 958  26.80  17  80.4  197 810  189 722  4.09  18  66.7  133 594  99 469  25.54  19  78.3  237 700  136 442  42.60  20  69  246 148  96 072  60.97  21  70.1  168 431  147 994  12.13  22  75.1  158 885  86 504  45.56  Mean Standard Error        0.003 13.93  Absolute Mean        41.95  Standard Error        10.50  View Large Figure 3. View largeDownload slide Estimated mean clutch size for different-sized female Panulirus argus using the non-invasive method and previously published studies for Mexico (Fonesca-Larios & Briones-Fourzán, 1998), Florida (Cox & Bertelsen, 1997), Brazil (Nascimento & Araújo, 1984), and Cuba (Cruz et al., 1987). Figure 3. View largeDownload slide Estimated mean clutch size for different-sized female Panulirus argus using the non-invasive method and previously published studies for Mexico (Fonesca-Larios & Briones-Fourzán, 1998), Florida (Cox & Bertelsen, 1997), Brazil (Nascimento & Araújo, 1984), and Cuba (Cruz et al., 1987). Currie et al. (2010) noted that the mean percent difference between traditional gravimetric estimates of fecundity and this non-invasive method was 3.68%, thus concluding that the method was reliable. Using the same approach as Currie et al. (2010) to calculate the mean difference between methods, our difference was even lower at 0.003%, suggesting that the non-invasive method could also be applicable for P. argus. If absolute values are used in the calculation the mean difference between the non-invasive and gravimetric methods was 42%, although this difference was still not statistically significant. Unfortunately, we know of no studies that simultaneously provided actual counts of each and every egg in a clutch, a tedious task when each clutch is comprised of tens to hundreds of thousands of eggs and many clutches must be counted. So at present, there is no way of determining which of the two techniques provides a more accurate estimate of clutch size. When used to estimate fecundity for P. argus from the Florida Keys, the non-invasive method produced estimates that were considerably lower than those previously published and derived from traditional methods. If the non-invasive method of estimating clutch size is indeed more accurate than the gravimetric method, then size-specific fecundity is lower in the Florida Keys than previously estimated there and at other locations. The lower estimates produced by the non-invasive method, however, could be influenced by the non-linear relationship between female size and clutch size and the small range of female sizes we used to generate the regression equation (equation 3). Previous research indicates that non-linearity in the regression equations becomes more apparent as female P. argus get larger than 90 mm CL (MacDiarmid & Sainte-Marie, 2006). Egg diameters can also vary relative to female size and clutch number where females produce multiple clutches in a year (GG, unpublished data), and it is possible that packing density differs as female lobsters grow (Koopman et al., 2015). If so, the non-invasive method would have to be validated for larger females before one could reliably estimate clutch size for lobsters greater than 90 mm CL. As Currie et al. (2010) noted, a slight error in the measurement of egg height can have a disproportionate influence on the total egg volume calculated. A change of ± 1 mm in average height can alter fecundity estimates by as much as ± 1,000 eggs lobster–1 (Currie et al., 2010). The estimated clutch size of individual 6 (Table 1) differed by 221% between methods. Careful measurements are therefore required for precise estimates of clutch size. Part of the challenge in using the non-invasive method for P. argus is the potentially different egg sizes and packing densities relative to egg development stage, clutch number, spawning time (i.e., early or late in the breeding season), and geographic location, all of which could influence egg mass volume and mean egg volume. Although we calculated an egg packing density correction of 0.4225 it may require alterations for females with eggs at different development stages or sizes. These potential confounding factors are rarely accounted for in studies estimating fecundity using traditional methods and, for the sake of consistency, we only used early-stage, first-clutch, bright-orange (i.e., no visible eyespots) eggs. The variability associated with the non-invasive method is regrettable because the method does offer some clear advantages over other methods: it is quick, inexpensive, and can easily be applied in the field. It only requires measurements of egg mass, length, and height in addition to the removal of a small subsample of eggs with which to calculate individual egg volume. In fisheries stock assessment, a single discrete study is often relied on for the estimates of fecundity that underpin the needed length-fecundity relationship. In these cases, the issue of destructive sampling is perhaps not problematic and gravimetric methods will suffice. In other research settings, however, destructive sampling is undesirable, as in field studies of changes in lobster fecundity through time or among regions as part of annual catch sampling to monitor potential effects of environmental change or sex ratio in the stock. Laboratory studies of lobster fecundity would also benefit from a non-invasive approach, as in cases where multiple clutches of each female must be examined. Non-invasive methods are also more consistent with laboratory animal welfare policies and more in keeping with conservation and management objectives. ACKNOWLEDGEMENTS The authors wish to thank those who assisted in the field and laboratory: E. Anderson, S. Hagedorn, C. McCoy, R. Ramos, M. Short, J. Spadaro, N. Slayden, and B. Weibe. We also thank two anonymous reviewers for their feedback on the manuscript. Funding was provided by grants to GG from Fulbright NZ, PEO, and Old Dominion University and by a NOAA-SK award (13SK044) to MB. Lobsters were collected under permits SAL-13-0582B-SRP and SAL-16-0582-SRP from the Florida Wildlife Conservation Commission and under a permit (FKNMS-2014–107) from the NOAA Florida Keys National Marine Sanctuary. REFERENCES Bryan, J.L.M., Wildhaber, M.L, Papoulias, D.M, DeLonay, A.J, Tillitt, D.E. & Annis, M.L. 2007. Estimation of gonad volume, fecundity, and reproductive stage of shovelnose sturgeon using sonography and endoscopy with application to the endangered pallid sturgeon. Journal of Applied Ichthyology , 23: 411– 419. Google Scholar CrossRef Search ADS   Chubb, C.F. 2008. Reproductive biology: issues for management. In: Spiny lobsters: fisheries and culture , edn. 2 ( B. Phillips & J. Kittaka, eds.), pp. 245– 276. Fishing News Books, Oxford, UK. Google Scholar CrossRef Search ADS   Cox, C. & Bertelsen, R.D. 1997. Fecundity of the Caribbean spiny lobster, Panulirus argus from fished and unfished regions in the Florida Keys, USA (abstract), pp. 24– 25. In: Fifth International Conference and Workshop on Lobster Biology and Management , Queenstown, New Zealand. CRFM (Caribbean Regional Fisheries Mechanism), 2011. Baseline review of the status and management of the Caribbean spiny lobster fisheries in the CARICOM region . CRFM Technical & Advisory Document 2011/5. Belize City, Belize. Cruz, R. & de León, M.E. 1991. Dinámica reproductiva de la langosta (Panulirus argus) en el archipiélago cubano. Revista de Investigaciones Marinas (La Habana) , 12: 234– 245. Cruz, R., Baisre, J.A, Díaz-Iglesias, E, Brito, R, García, C, Blanco, W. & Carrodeguas, C. 1987. Atlas biológico-pesquero de la langosta en el archipiélago cubano . Centro de Investigaciones Pesqueras, La Habana, Cuba. Currie, J.J., Schneider, D.C. & Wilke, K.M. 2010. Validation of a noninvasive technique for estimating fecundity in the American lobster Homarus americanus. Journal of Shellfish Research , 29: 1021– 1024. Google Scholar CrossRef Search ADS   Diaz, H., Conde, J.E. & Bevilacqua, M. 1983. A volumetric method for estimating fecundity in Decapoda. Marine Ecology Progress Series , 10: 203– 206. Google Scholar CrossRef Search ADS   Ehrhardt, N.M., Puga, R. & Butler, M.J. IV. 2010. Large ecosystem dynamics and fishery management concepts: The Caribbean spiny lobster, Panulirus argus, fisheries. In: Towards marine ecosystem-based management in the wider Caribbean  ( L. Fanning, R. Mahon & P. McConney, eds.), Amsterdam University Press, Amsterdam. FAO (Food and Agriculture Organization of the United Nations). 1974. Part 2: Methods of resource investigation and their application. In: Manual of fisheries science  ( M.J. Holden & D.F.S Raitt, eds.). FAO, Rome. Fonseca-Larios, M.E. & Briones-Fourzán, P. 1998. Fecundity of the spiny lobster Panulirus argus (Latreille, 1804) in the Caribbean coast of Mexico. Bulletin of Marine Science , 63: 21– 32. Gotelli, N.J. 2008. A primer of ecology , edn. 4. Sinauer, Sunderland, MA, USA. Koopman, H.N., Westgate, A.J. & Siders, Z.A. 2015. Declining fecundity and factors affecting embryo quality in the American lobster (Homarus americanus) from the Bay of Fundy. Canadian Journal of Fisheries and Aquatic Sciences , 72: 352– 363. Google Scholar CrossRef Search ADS   Latreille, P.A. 1804. Tableau Méthodique des Crustacés. In: Nouveau dictionnaire d’Histoire Naturelle, appliquée aux arts, principalement à l’agriculture et à l’économie rurale et domestique, par une Société de Naturalistes et d’Agriculteurs, avec des figures tirées des trois Règnes de la Nature . Vol. 24. Paris. MacDiarmid, A.B. & Sainte-Marie, B. 2006. Reproduction. In: Lobsters: biology, management, aquaculture and fisheries  ( B. Phillips, ed.), pp 45– 77. Blackwell, Oxford, UK. Google Scholar CrossRef Search ADS   Milne Edwards, H. 1837. Histoire naturelle des Crustacés, comprenant l’anatomie, la physiologie et la classification de ces animaux , Vol. 2. Librairie Encyclopédique de Roret, Paris. Nascimento, I.V. & Araújo, M.E. 1984. Fecundidade das lagostas Panulirus argus e Panulirus laevicauda (Latri) capturadas no litoral do Rio Grande do Norde. Estudos de Pesca (Recife) , 11: 39– 42. © The Author(s) 2017. Published by Oxford University Press on behalf of The Crustacean Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Crustacean Biology Oxford University Press

Application of a non-invasive technique for estimating clutch size in the Caribbean spiny lobster Panulirus argus (Latreille, 1804)

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Abstract

Abstract We investigated the use of a non-invasive method of fecundity estimation for the Caribbean spiny lobster, Panulirus argus (Latreille, 1804), using a method previously described for the American clawed lobster, Homarus americanus (Milne Edwards, 1837). This method only requires the removal of a small subsample of eggs from each individual as compared to traditional gravimetric methods that strip females of their entire clutch. Comparisons of the non-invasive method with traditional estimates resulted in egg count estimates that differed by a mean of 0.003% but a mean absolute value of 42%. Our results demonstrate the utility of this rapid, inexpensive, and non-destructive method for estimating clutch size for a highly fecund species. Differences between estimates produced using this new method and the traditional gravimetric method are substantial, and therefore it’s accuracy in relation to the traditional gravimetric method remains unknown. Fecundity is a key parameter in population ecology, conservation biology, and the setting of biological references points in fisheries stock assessment (FAO, 1974; Gotelli, 2008). Estimates of fecundity, particularly in marine fishes and invertebrates, has traditionally employed invasive and oft-times destructive methods. Gravimetric estimation methods are most common and involve removing and drying egg masses, counting the number of eggs in a weighed subsample, and dividing the total weight of the egg mass by the mean weight of a single egg (Diaz et al., 1983; Chubb, 2008). Methods that destroy the entire clutch or that require the death of the female, however, preclude the potential for further study of egg and larval development and runs counter to conservation or management measures that aim to maintain spawning stock or promote reproduction. Non-invasive techniques to estimate fecundity using sonography, endoscopy, and photography with image analysis for externally extruded clutches have been attempted in a number of species (Bryan et al., 2007) but these methods can be cost prohibitive, require specialized equipment, or are time consuming. We investigated the use of a non-invasive method to estimate clutch size in the Caribbean spiny lobster, Panulirus argus (Latreille,1804), using the method devised by Currie et al. (2010) for the American lobster, Homarus americanus (Milne Edwards, 1837). The Caribbean spiny lobster is an iconic and economically valuable species; it sustains the primary fishery for 24 Caribbean nations and employs an estimated 50,000 fishers and an additional 200,000 in fishery -related jobs (CRFM, 2011). As a consequence of their high value and market demand, however, many populations of P. argus are currently fully capitalized or overfished (Ehrhardt et al., 2010). Size-fecundity relationships of P. argus have been well studied and there are estimates for populations throughout its range including Brazil (Nascimento & Araújo, 1984), Cuba (Cruz et al., 1987), Florida (Cox & Bertelsen, 1997), and Mexico (Fonseca-Larios & Briones-Fourzán, 1998). Female P. argus larger than 100 mm carapace length (CL) are highly fecund, carrying an estimated hundreds of thousands of eggs in a single clutch (Cox & Bertelsen, 1997). Methods that rely on counting every egg in a clutch are thus impractical for P. argus and other highly fecund species, thus most investigators rely on gravimetric estimations. These methods are still invasive and require not only the removal of the clutch from the female, but also the pleopodal setae to which the eggs are attached. For species like P. argus that produce multiple clutches in a single reproductive season, doing so prohibits the attachment of eggs in subsequent clutches until the female molts again into a reproductive condition. Egg removal thus dooms the reproductive success of this individual for months to years, depending on the reproductive and molting dynamics of the species. Moreover, we are unaware of any study using a gravimetric-based fecundity estimate for lobsters that also included counts of all the eggs in a clutch. The accuracy of this commonly used approach is thus unknown for these highly fecund species. In comparison, the non-invasive method used by Currie et al. (2010) offers a means of estimating clutch size quickly and without specialized equipment, and without the need to remove the entire clutch from an individual, thus allowing the remaining eggs to continue to develop and the females to continue normal reproductive activities. Twenty-two ovigerous females ranging in size from 65.4 to 90.2 mm CL were collected by hand by divers in the Florida Keys, Florida, USA in July 2016 and 2017. Only females with eggs that were bright orange in color (i.e., within the first 1.5 weeks of spawning) were selected. The arrangement of the endopodites (the inner portion of the pleopods) in female P. argus, splits the egg mass into segments (Fig. 1). Larger females typically have four segments, whereas smaller females have three. The length of the entire egg mass and the height of each egg segment was measured using a ruler (Fig. 1). For each egg segment, the ruler was inserted into the center of the egg mass between each segment until it touched the abdomen surface. Figure 1. View largeDownload slide Measurements taken to estimate fecundity: length and height of segments. This figure is available in color at Journal of Crustacean Biology online. Figure 1. View largeDownload slide Measurements taken to estimate fecundity: length and height of segments. This figure is available in color at Journal of Crustacean Biology online. The volume of the entire egg mass was then calculated using the formula for the volume of a cylinder:  volume of egg mass=(πH2L2)×0.4225 (1) where H is the average height of the egg segments and L is the length of the entire egg mass. The volume × length equation is divided by 2 to account for the fact that only half the cylinder contains eggs (i.e., eggs are carried externally). Volume is then multiplied by 0.4225, which is the calculated egg packing density (see below). To calculate mean egg volume, at least 10 eggs were removed from each female and stored in 20 ml scintillation vials containing a 5% formalin-seawater solution. The longest and shortest axes of 10 eggs were then measured under a compound microscope (40× magnification) and averaged to provide an average egg radius. Mean egg volume was thus calculated as:  egg volume=43(πr3) (2) where r is the egg radius. Clutch size was then calculated by dividing the volume of the entire egg mass (equation 1) by mean egg volume (equation 2). To validate estimates of egg counts using the non-invasive method, the entire clutch was removed from the 22 females and preserved in a 5% formalin-seawater solution for 24 h. Eggs were rinsed in freshwater and dried at 60 °C for 48 h. Dried eggs were gently sifted through a 300 μm mesh sieve to remove the funiculae, the connective tissue that attaches eggs to the setae. The eggs were then weighed and five weighed subsamples (of greater than 30 eggs sample–1) were counted under a microscope. Clutch size was calculated as total clutch weight divided by mean average egg weight. A paired t-test was used to compare non-invasive and invasive techniques, with a null hypothesis of no difference in calculated clutch size (R version 3.3.1). The non-invasive method was applied to lobsters caught in the Florida Keys during the summer of 2015 and 2016. The length of egg masses and heights of segment were measured in 102 females (63 to 141 mm CL) with bright orange eggs. The mean egg volume from the 22 sampled lobsters was used in the fecundity estimates, and calculations for clutch size were plotted against female CL. As the relationship between CL and fecundity is typically non-linear, particularly as females get larger, we plotted clutch size relative to female CL (in mm) following log transformation, to obtain the following equation (R2 = 0.7805; standard error = intercept 0.304; slope 0.1520) (Fig. 2).  Log clutch size = 2.8669 log CL–0.3442 (3) Figure 2. View largeDownload slide Estimated mean clutch size (non-invasive method) and carapace length (log transformed) for 102 female Panulirus argus (63–141 mm carapace length) sampled from the Florida Keys, FL. Figure 2. View largeDownload slide Estimated mean clutch size (non-invasive method) and carapace length (log transformed) for 102 female Panulirus argus (63–141 mm carapace length) sampled from the Florida Keys, FL. Back-transformed estimates of clutch size were then compared with estimates from equations for P. argus in the Caribbean: Brazil (Nascimento & Araújo, 1984), Cuba (Cruz et al., 1987), Florida (Cox & Bertelsen, 1997), and Mexico (Fonseca-Larios & Briones-Fourzán, 1998). A reduction in the egg mass volume of 42.25% was incorporated into the egg mass equation to account for egg packing density and to reduce the difference in estimates between the traditional and non-invasive methods. This value was calculated by reducing the density of the egg mass volume by 1% until the smallest percent difference between the traditional method and non-invasive method was found. With this correction, clutch size estimates using the non-invasive method were, on average, only 0.003% lower than gravimetric estimates with a standard error of 13.93% (Table 1). The average difference was nevertheless 42% with a standard error of 10.50% (Table 1) when calculated with absolute values. The mean differences in clutch size estimates between our method and gravimetric-based estimates, however, did not differ significantly (paired t-test, t = 1.3655, df = 21, P = 0.1865). As compared to previously published equations from other locations in the Caribbean, our regression equation produced estimates of clutch size that were markedly lower, a mean percent difference of 62% from the next closest values from Brazil (Fig. 3). Table 1. Fecundity estimates of Panulirus argus using non-invasive and traditional methods. Lobster ID  Lobster carapace length (mm)  Traditional method  Non-invasive method  % Difference  1  84.2  224 712  123 147  45.19  2  82.7  140 329  165 267  –17.77  3  79.5  146 033  218 651  –49.73  4  83.8  192 651  170 754  11.37  5  72.9  90 305  213 144  –136.02  6  65.4  16 498  52 981  –221.14  7  77.6  195 021  206 992  –6.14  8  73  148 434  89 035  40.02  9  83.2  233 377  250 854  –7.49  10  90.2  397 283  489 089  –23.11  11  65.7  181 894  75 559  58.46  12  70.8  142 782  86 453  39.45  13  70.7  88 462  81 357  8.03  14  62  84 650  64 462  23.85  15  56.2  48 940  40 417  17.42  16  75.5  139 292  101 958  26.80  17  80.4  197 810  189 722  4.09  18  66.7  133 594  99 469  25.54  19  78.3  237 700  136 442  42.60  20  69  246 148  96 072  60.97  21  70.1  168 431  147 994  12.13  22  75.1  158 885  86 504  45.56  Mean Standard Error        0.003 13.93  Absolute Mean        41.95  Standard Error        10.50  Lobster ID  Lobster carapace length (mm)  Traditional method  Non-invasive method  % Difference  1  84.2  224 712  123 147  45.19  2  82.7  140 329  165 267  –17.77  3  79.5  146 033  218 651  –49.73  4  83.8  192 651  170 754  11.37  5  72.9  90 305  213 144  –136.02  6  65.4  16 498  52 981  –221.14  7  77.6  195 021  206 992  –6.14  8  73  148 434  89 035  40.02  9  83.2  233 377  250 854  –7.49  10  90.2  397 283  489 089  –23.11  11  65.7  181 894  75 559  58.46  12  70.8  142 782  86 453  39.45  13  70.7  88 462  81 357  8.03  14  62  84 650  64 462  23.85  15  56.2  48 940  40 417  17.42  16  75.5  139 292  101 958  26.80  17  80.4  197 810  189 722  4.09  18  66.7  133 594  99 469  25.54  19  78.3  237 700  136 442  42.60  20  69  246 148  96 072  60.97  21  70.1  168 431  147 994  12.13  22  75.1  158 885  86 504  45.56  Mean Standard Error        0.003 13.93  Absolute Mean        41.95  Standard Error        10.50  View Large Table 1. Fecundity estimates of Panulirus argus using non-invasive and traditional methods. Lobster ID  Lobster carapace length (mm)  Traditional method  Non-invasive method  % Difference  1  84.2  224 712  123 147  45.19  2  82.7  140 329  165 267  –17.77  3  79.5  146 033  218 651  –49.73  4  83.8  192 651  170 754  11.37  5  72.9  90 305  213 144  –136.02  6  65.4  16 498  52 981  –221.14  7  77.6  195 021  206 992  –6.14  8  73  148 434  89 035  40.02  9  83.2  233 377  250 854  –7.49  10  90.2  397 283  489 089  –23.11  11  65.7  181 894  75 559  58.46  12  70.8  142 782  86 453  39.45  13  70.7  88 462  81 357  8.03  14  62  84 650  64 462  23.85  15  56.2  48 940  40 417  17.42  16  75.5  139 292  101 958  26.80  17  80.4  197 810  189 722  4.09  18  66.7  133 594  99 469  25.54  19  78.3  237 700  136 442  42.60  20  69  246 148  96 072  60.97  21  70.1  168 431  147 994  12.13  22  75.1  158 885  86 504  45.56  Mean Standard Error        0.003 13.93  Absolute Mean        41.95  Standard Error        10.50  Lobster ID  Lobster carapace length (mm)  Traditional method  Non-invasive method  % Difference  1  84.2  224 712  123 147  45.19  2  82.7  140 329  165 267  –17.77  3  79.5  146 033  218 651  –49.73  4  83.8  192 651  170 754  11.37  5  72.9  90 305  213 144  –136.02  6  65.4  16 498  52 981  –221.14  7  77.6  195 021  206 992  –6.14  8  73  148 434  89 035  40.02  9  83.2  233 377  250 854  –7.49  10  90.2  397 283  489 089  –23.11  11  65.7  181 894  75 559  58.46  12  70.8  142 782  86 453  39.45  13  70.7  88 462  81 357  8.03  14  62  84 650  64 462  23.85  15  56.2  48 940  40 417  17.42  16  75.5  139 292  101 958  26.80  17  80.4  197 810  189 722  4.09  18  66.7  133 594  99 469  25.54  19  78.3  237 700  136 442  42.60  20  69  246 148  96 072  60.97  21  70.1  168 431  147 994  12.13  22  75.1  158 885  86 504  45.56  Mean Standard Error        0.003 13.93  Absolute Mean        41.95  Standard Error        10.50  View Large Figure 3. View largeDownload slide Estimated mean clutch size for different-sized female Panulirus argus using the non-invasive method and previously published studies for Mexico (Fonesca-Larios & Briones-Fourzán, 1998), Florida (Cox & Bertelsen, 1997), Brazil (Nascimento & Araújo, 1984), and Cuba (Cruz et al., 1987). Figure 3. View largeDownload slide Estimated mean clutch size for different-sized female Panulirus argus using the non-invasive method and previously published studies for Mexico (Fonesca-Larios & Briones-Fourzán, 1998), Florida (Cox & Bertelsen, 1997), Brazil (Nascimento & Araújo, 1984), and Cuba (Cruz et al., 1987). Currie et al. (2010) noted that the mean percent difference between traditional gravimetric estimates of fecundity and this non-invasive method was 3.68%, thus concluding that the method was reliable. Using the same approach as Currie et al. (2010) to calculate the mean difference between methods, our difference was even lower at 0.003%, suggesting that the non-invasive method could also be applicable for P. argus. If absolute values are used in the calculation the mean difference between the non-invasive and gravimetric methods was 42%, although this difference was still not statistically significant. Unfortunately, we know of no studies that simultaneously provided actual counts of each and every egg in a clutch, a tedious task when each clutch is comprised of tens to hundreds of thousands of eggs and many clutches must be counted. So at present, there is no way of determining which of the two techniques provides a more accurate estimate of clutch size. When used to estimate fecundity for P. argus from the Florida Keys, the non-invasive method produced estimates that were considerably lower than those previously published and derived from traditional methods. If the non-invasive method of estimating clutch size is indeed more accurate than the gravimetric method, then size-specific fecundity is lower in the Florida Keys than previously estimated there and at other locations. The lower estimates produced by the non-invasive method, however, could be influenced by the non-linear relationship between female size and clutch size and the small range of female sizes we used to generate the regression equation (equation 3). Previous research indicates that non-linearity in the regression equations becomes more apparent as female P. argus get larger than 90 mm CL (MacDiarmid & Sainte-Marie, 2006). Egg diameters can also vary relative to female size and clutch number where females produce multiple clutches in a year (GG, unpublished data), and it is possible that packing density differs as female lobsters grow (Koopman et al., 2015). If so, the non-invasive method would have to be validated for larger females before one could reliably estimate clutch size for lobsters greater than 90 mm CL. As Currie et al. (2010) noted, a slight error in the measurement of egg height can have a disproportionate influence on the total egg volume calculated. A change of ± 1 mm in average height can alter fecundity estimates by as much as ± 1,000 eggs lobster–1 (Currie et al., 2010). The estimated clutch size of individual 6 (Table 1) differed by 221% between methods. Careful measurements are therefore required for precise estimates of clutch size. Part of the challenge in using the non-invasive method for P. argus is the potentially different egg sizes and packing densities relative to egg development stage, clutch number, spawning time (i.e., early or late in the breeding season), and geographic location, all of which could influence egg mass volume and mean egg volume. Although we calculated an egg packing density correction of 0.4225 it may require alterations for females with eggs at different development stages or sizes. These potential confounding factors are rarely accounted for in studies estimating fecundity using traditional methods and, for the sake of consistency, we only used early-stage, first-clutch, bright-orange (i.e., no visible eyespots) eggs. The variability associated with the non-invasive method is regrettable because the method does offer some clear advantages over other methods: it is quick, inexpensive, and can easily be applied in the field. It only requires measurements of egg mass, length, and height in addition to the removal of a small subsample of eggs with which to calculate individual egg volume. In fisheries stock assessment, a single discrete study is often relied on for the estimates of fecundity that underpin the needed length-fecundity relationship. In these cases, the issue of destructive sampling is perhaps not problematic and gravimetric methods will suffice. In other research settings, however, destructive sampling is undesirable, as in field studies of changes in lobster fecundity through time or among regions as part of annual catch sampling to monitor potential effects of environmental change or sex ratio in the stock. Laboratory studies of lobster fecundity would also benefit from a non-invasive approach, as in cases where multiple clutches of each female must be examined. Non-invasive methods are also more consistent with laboratory animal welfare policies and more in keeping with conservation and management objectives. ACKNOWLEDGEMENTS The authors wish to thank those who assisted in the field and laboratory: E. Anderson, S. Hagedorn, C. McCoy, R. Ramos, M. Short, J. Spadaro, N. Slayden, and B. Weibe. We also thank two anonymous reviewers for their feedback on the manuscript. Funding was provided by grants to GG from Fulbright NZ, PEO, and Old Dominion University and by a NOAA-SK award (13SK044) to MB. Lobsters were collected under permits SAL-13-0582B-SRP and SAL-16-0582-SRP from the Florida Wildlife Conservation Commission and under a permit (FKNMS-2014–107) from the NOAA Florida Keys National Marine Sanctuary. REFERENCES Bryan, J.L.M., Wildhaber, M.L, Papoulias, D.M, DeLonay, A.J, Tillitt, D.E. & Annis, M.L. 2007. Estimation of gonad volume, fecundity, and reproductive stage of shovelnose sturgeon using sonography and endoscopy with application to the endangered pallid sturgeon. Journal of Applied Ichthyology , 23: 411– 419. Google Scholar CrossRef Search ADS   Chubb, C.F. 2008. Reproductive biology: issues for management. In: Spiny lobsters: fisheries and culture , edn. 2 ( B. Phillips & J. Kittaka, eds.), pp. 245– 276. Fishing News Books, Oxford, UK. Google Scholar CrossRef Search ADS   Cox, C. & Bertelsen, R.D. 1997. Fecundity of the Caribbean spiny lobster, Panulirus argus from fished and unfished regions in the Florida Keys, USA (abstract), pp. 24– 25. In: Fifth International Conference and Workshop on Lobster Biology and Management , Queenstown, New Zealand. CRFM (Caribbean Regional Fisheries Mechanism), 2011. Baseline review of the status and management of the Caribbean spiny lobster fisheries in the CARICOM region . CRFM Technical & Advisory Document 2011/5. Belize City, Belize. Cruz, R. & de León, M.E. 1991. Dinámica reproductiva de la langosta (Panulirus argus) en el archipiélago cubano. Revista de Investigaciones Marinas (La Habana) , 12: 234– 245. Cruz, R., Baisre, J.A, Díaz-Iglesias, E, Brito, R, García, C, Blanco, W. & Carrodeguas, C. 1987. Atlas biológico-pesquero de la langosta en el archipiélago cubano . Centro de Investigaciones Pesqueras, La Habana, Cuba. Currie, J.J., Schneider, D.C. & Wilke, K.M. 2010. Validation of a noninvasive technique for estimating fecundity in the American lobster Homarus americanus. Journal of Shellfish Research , 29: 1021– 1024. Google Scholar CrossRef Search ADS   Diaz, H., Conde, J.E. & Bevilacqua, M. 1983. A volumetric method for estimating fecundity in Decapoda. Marine Ecology Progress Series , 10: 203– 206. Google Scholar CrossRef Search ADS   Ehrhardt, N.M., Puga, R. & Butler, M.J. IV. 2010. Large ecosystem dynamics and fishery management concepts: The Caribbean spiny lobster, Panulirus argus, fisheries. In: Towards marine ecosystem-based management in the wider Caribbean  ( L. Fanning, R. Mahon & P. McConney, eds.), Amsterdam University Press, Amsterdam. FAO (Food and Agriculture Organization of the United Nations). 1974. Part 2: Methods of resource investigation and their application. In: Manual of fisheries science  ( M.J. Holden & D.F.S Raitt, eds.). FAO, Rome. Fonseca-Larios, M.E. & Briones-Fourzán, P. 1998. Fecundity of the spiny lobster Panulirus argus (Latreille, 1804) in the Caribbean coast of Mexico. Bulletin of Marine Science , 63: 21– 32. Gotelli, N.J. 2008. A primer of ecology , edn. 4. Sinauer, Sunderland, MA, USA. Koopman, H.N., Westgate, A.J. & Siders, Z.A. 2015. Declining fecundity and factors affecting embryo quality in the American lobster (Homarus americanus) from the Bay of Fundy. Canadian Journal of Fisheries and Aquatic Sciences , 72: 352– 363. Google Scholar CrossRef Search ADS   Latreille, P.A. 1804. Tableau Méthodique des Crustacés. In: Nouveau dictionnaire d’Histoire Naturelle, appliquée aux arts, principalement à l’agriculture et à l’économie rurale et domestique, par une Société de Naturalistes et d’Agriculteurs, avec des figures tirées des trois Règnes de la Nature . Vol. 24. Paris. MacDiarmid, A.B. & Sainte-Marie, B. 2006. Reproduction. In: Lobsters: biology, management, aquaculture and fisheries  ( B. Phillips, ed.), pp 45– 77. Blackwell, Oxford, UK. Google Scholar CrossRef Search ADS   Milne Edwards, H. 1837. Histoire naturelle des Crustacés, comprenant l’anatomie, la physiologie et la classification de ces animaux , Vol. 2. Librairie Encyclopédique de Roret, Paris. Nascimento, I.V. & Araújo, M.E. 1984. Fecundidade das lagostas Panulirus argus e Panulirus laevicauda (Latri) capturadas no litoral do Rio Grande do Norde. Estudos de Pesca (Recife) , 11: 39– 42. © The Author(s) 2017. Published by Oxford University Press on behalf of The Crustacean Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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The Journal of Crustacean BiologyOxford University Press

Published: Jan 1, 2018

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