North American Journal of Fisheries Management 28:442–446, 2008 Ó Copyright by the American Fisheries Society 2008 DOI: 10.1577/M07-115.1
[Management Brief]
Validation of Daily Growth Increment Formation in the Otoliths of Juvenile Cyprinid Fishes from the Brazos River, Texas BART W. DURHAM*
AND
GENE R. WILDE
Department of Biological Sciences, Texas Tech University, Lubbock, Texas 79409-3131, USA Abstract.—Age data are commonly used by fisheries biologists to assess a number of important population characteristics. For cyprinid fishes, there have been few attempts to assess the validity of age estimates, particularly those based on otolith microstructure. We assessed the periodicity of growth increment formation in the otoliths of three species of cyprinid from the Brazos River, Texas, two of which are of conservation concern. We immersed juvenile sharpnose shiners Notropis oxyrhynchus, smalleye shiners N. buccula, and plains minnow Hybognathus placitus in a 100mg/L solution of alizarin complexone for 24 h and sampled fish at 5-d intervals for 30 d. Regression models indicated high correspondence between number of days posttreatment and number of growth increments between the alizarin mark and the edge of the otolith for all three species (sharpnose shiner: r2 ¼ 0.99; smalleye shiner: r2 ¼ 0.98; plains minnow: r2 ¼ 0.97). Slopes of regression lines did not differ from 1.0 (t-test: P , 0.05), indicating that growth increments are deposited daily in these three species. Our results provide evidence that daily growth increments are reliable sources of age information for cyprinids.
Population dynamics of fishes are greatly influenced by early life history events (e.g., May 1974; Kamler 1992; Thorisson 1994; Kelso and Rutherford 1996), and recent studies have shown that population growth rates of many North American cyprinid species ultimately are determined by the dynamics of the juvenile life stage (Ve´lez-Espino et al. 2006; Durham 2007). Thus, a precise understanding of the factors that affect early life history of cyprinids is critical for successful management and conservation, especially as we are faced with an increasing number of imperiled species (Williams et al. 1989; Johnston 1999; Nguyen and De Silva 2006). The discovery of daily growth increments in otoliths of young fishes (Panella 1971) was important because it provided researchers with a tool for determining a number of important early life history characteristics associated with age and growth of juvenile fish, such as reproductive and recruitment patterns, hatch dates, settlement dates, and growth and mortality rates (e.g., Miller and Stork 1984; Essig and Cole 1986; Isely and Noble 1987). However, proper * Corresponding author:
[email protected] Received February 6, 2007; accepted July 16, 2007 Published online March 13, 2008
use of age and growth data from otoliths requires that the periodicity of growth increment formation be validated (Campana and Neilson 1985). This requirement has been frequently overlooked or ignored (Beamish and McFarlane 1983; Campana 2001). Most validation studies have been conducted on commercially or recreationally important species, whereas nongame and commercially unimportant species have received relatively little attention. For cyprinid species in particular, validation studies have been infrequently conducted despite the fact that this family is the most speciose among fishes. Age validation has been conducted for only 10 cyprinid species. Annual ring formation has been validated in the otoliths of common carp Cyprinus carpio (Vilizzi and Walker 1999), roundtail chub Gila robusta (Brouder 2005), fallfish Semotilus corporalis (Victor and Brothers 1982), Utah chub G. atraria (Johnson and Belk 2004), and Sclater’s barbel Barbus sclateri (Escot and Granado-Lorencio 2001). Daily growth increments have been validated for only eight species: common carp (Smith and Walker 2003), roundtail chub (Brouder 2005), fallfish (Victor and Brothers 1982), northern pikeminnow Ptychocheilus oregonensis (Wertheimer and Barfoot 1998), splittail Pogonichthys macrolepidotus (Feyrer et al. 2004), mukene Rastrineobola argentea (Njiru et al. 2001), sanjika Opsaridium microcephalum (Morioka and Matsumoto 2003), and dwarf sanjika O. tweddleorum (Morioka and Matsumoto 2007). Age validation is most reliable when otoliths are obtained from fish of known age; however, the difficulty and cost associated with the husbandry of many species makes using known-age fish impractical. In these cases, a chemical reference mark can be induced in the otolith by exposing fish to a chemical fluorochrome marker by immersion, injection, or ingestion. The antibiotic oxytetracycline has been the most common chemical marker used in age validation studies, but alizarin compounds have recently shown promise as an alternative to oxytetracycline (Tsukamoto 1988; Szedlmayer and Howe 1995; Thomas et al. 1995; Beckman and Schulz 1996). Alizarin compounds may be preferable to oxytetracycline for several reasons. First, visible marks have been achieved with
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low concentrations of alizarin, which reduces the potential for mortality during and after immersion (van der Walt and Faragher 2003). Alizarin marks often can be detected without the aid of a fluorescent light source (Beckman and Schulz 1996; Hoff et al. 1997). Treatment with alizarin results in a bright-red reference mark, whereas treatment with oxytetracycline typically results in a yellow or dull green that can be difficult to see clearly on fish otoliths. Finally, degradation of marks by exposure to sunlight, as occurs for oxytetracycline (e.g., Lorson and Mudrak 1987), has not been reported for alizarin. The realization that the juvenile life stage so heavily influences overall population growth of cyprinids (Ve´lez-Espino et al. 2006), along with the growing number of imperiled species for which conservation efforts may be required, hastens the need for accurate information on age and growth of this taxon. This study reports the results of an experiment in which alizarin complexone was used to validate the formation of daily growth increments in the otoliths of three cyprinid species from the upper Brazos River, Texas, two of which are candidates for protection under the U.S. Endangered Species Act. Methods Juvenile fishes were collected from two sites on the Brazos River during August 2003 and transported to an aquatic research laboratory at Texas Tech University, Lubbock. To avoid unnecessary stress to the fish, we made no attempt to identify individuals to species in the field. However, before specimens were collected for transport, a preliminary sample was inspected in the field to ensure that juveniles of multiple species were present at the sampling locations. Four cyprinid species were sampled, including the sharpnose shiner Notropis oxyrhynchus, smalleye shiner N. buccula, plains minnow Hybognathus placitus, and shoal chub Macrhybopsis hyostoma. Fish were collected with a smallmesh seine (1.8 3 3.4 m; 1-mm mesh) from multiple microhabitats within each site to increase the chances of collecting a sufficient number of all species present. In the laboratory, fish were placed in an aerated, 29-L aquarium and allowed to acclimate to laboratory conditions for 2 weeks. Natural photoperiod was mimicked in the laboratory with timer-controlled lights. Laboratory temperature ranged between 178C and 198C for the duration of the trial. Fish were fed a combination of commercial flake food, freeze-dried brine shrimp Artemia spp., and frozen bloodworms (Chironomidae) ad libitum twice per day. Uneaten food and solid waste were removed with a siphon after the second feeding of the day. Routine water exchanges
were performed as necessary to maintain low ammonia levels. After the acclimation period, fish were transferred to an aquarium containing dissolved alizarin complexone (ICN Biomedicals, Inc., Costa Mesa, California) at a concentration of 100 mg/L for 24 h (Tsukamoto 1988; Thomas et al. 1995; Beckman and Schulz 1996; Sugeha et al. 2001). Fish were then returned to the original aquarium. Water exchanges were performed every 8 h for the next 24 h because of leaching of residual alizarin from the fish tissue. Fish were sampled from the aquarium at 5-d intervals for the next 30 d, measured for total length, and preserved in a 95% solution of ethanol. To ensure that sufficient numbers of individuals would be available during the entire 30-d trial and because living fish of similar species were not easily identifiable, an attempt was made to sample at least 10 individuals of each species on each date while being careful to not sacrifice excessive numbers of any one species. Lapilli otoliths were removed and mounted on glass slides with thermoplastic cement. Daily growth increments were counted independently by two experienced readers using an inverted compound light microscope at 1003 magnification. A small number of the otoliths required polishing with alumina slurry and a polishing cloth to make growth increments more visible. Each reader counted both the total number of growth rings and the number of rings distal to the alizarin mark. When counts by the two readers were within 10% of one another, the final count was calculated as the mean of the two readings. If counts by the two readers varied by more than 10%, growth increments for that otolith were recounted by both readers. All otoliths for which readings could not be reconciled to within 10% were excluded from analyses. Linear regression was used to assess the degree of correspondence between the number of growth increments outside the alizarin mark and the number of days posttreatment. To determine whether increment formation occurred daily in each species, a t-test was used to assess the null hypothesis that the slope of the regression line did not differ from 1.0 (a ¼ 0.05; df ¼ n 2). Results Immersion of larval fish in 100-mg/L alizarin complexone for 24 h resulted in low mortality (,3% during treatment and for 72 h posttreatment). The mortality rates we observed were consistent with those reported in other marking studies using the same alizarin concentration (Tsukamoto 1988; Thomas et al. 1995; Beckman and Schulz 1996). In total, 171 individuals treated with alizarin complexone were
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TABLE 1.—Sample size, alizarin complexone marking efficiency, and range of otolith growth increments for three Brazos River (Texas) cyprinid species sampled during a growth increment validation trial, 2003. Species
N
Surface mark (%)
Embedded mark (%)
Range of increments
Sharpnose shiner Smalleye shiner Plains minnow
65 64 42
100 100 100
95 100 74
27–68 21–67 39–75
sampled and their daily growth increments were enumerated. The overall field sample contained sufficient numbers of smalleye shiners, sharpnose shiners, and plains minnow for validation of daily growth increments. Although shoal chub were common in the preliminary sample, the sample that was returned to the laboratory contained only one specimen. An initial inspection of otolith mounts with the naked eye and under transmitted light at 103 magnification (dissecting microscope) revealed that the alizarin complexone immersion had induced a scarlet coloration on the surface of all 171 otoliths. However, when growth increments were read under transmitted light with a compound microscope, clear alizarin marks embedded within the rings were not detectable in all otoliths. Alizarin marks were readily detectable in otoliths from smalleye and sharpnose shiners, but embedded marks could not be detected in 26% of otoliths from plains minnow (Table 1). Otoliths lacking marks that were visible under transmitted light were viewed under an ultraviolet light source to confirm the absence of an embedded alizarin mark, and no marks were detected. Regression models indicated a strong correspondence between number of rings distal to the alizarin mark and number of days posttreatment (Figure 1). Coefficients of determination (r2) for sharpnose shiner, smalleye shiner, and plains minnow regressions were extremely high (r2 ¼ 0.99, 0.98, and 0.97, respectively). For all three species, t-tests revealed that the slope of the regression line was not different from 1.0 (P , 0.05), indicating that growth increments were deposited daily. Variation in aging accuracy appeared to be similar on each sampling date except for plains minnow, which showed a slight increase in variation of aging accuracy on the 25- and 30-d samples (Figure 1). This result also was suggested by inspection of residual scatterplots for regression models. This variation in aging accuracy could be a reflection of normal variability in our particular sample or a result of difficulty associated with reading growth increments (reader error) at margins of otoliths from older plains minnow within
FIGURE 1.—Relationship between number of increments distal to the alizarin complexone mark on otoliths and number of days posttreatment for sharpnose shiners, smalleye shiners, and plains minnow collected from the Brazos River, Texas, in 2003. To allow visualization of all observations, data were jittered along the x-axis by adding a uniform random number between 1.0 and 1.0 to each observation. Regression lines and equations are based on unjittered data.
the sample (Table 1). However, scatterplots of the total number of growth increments and the difference between expected and observed increment counts for each fish showed that aging accuracy did not increase as total age of fish increased (this was true for all species, including plains minnow). Alternatively, the observed variation in aging accuracy for plains minnow could indicate slight nondaily increment deposition in fish that have attained a certain age. Regardless, variation in aging accuracy for plains minnow, even in the 25- and 30-d samples, was so slight that it did not
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cause the regression slope to deviate from 1.0, which suggests that across the range of ages (d) observed in our study, growth increments in juvenile plains minnow were deposited daily. Discussion Periodicity of growth increment formation in the otoliths of juvenile sharpnose shiners, smalleye shiners, and plains minnow was validated with alizarin complexone. Beckman and Schulz (1996) first reported that alizarin compounds could be used to induce marks in the otoliths of juvenile cyprinids; however, they did not validate the periodicity of increment formation for their study species. When combined, this study and the Beckman and Schulz (1996) study show that the technique is useful for validating daily growth increment formation for juvenile cyprinids when known-age fish cannot be used. Various combinations of chemical concentrations and immersion times for alizarin complexone have been suggested as optimal for inducing visible marks while maintaining low treatment and posttreatment mortality (e.g., Tsukamoto 1988; Thomas et al. 1995; Beckman and Schulz 1996; van der Walt and Faragher 2003). Although the optimum combination of chemical concentration and immersion time may indeed vary among species and life stages, the 100-mg/L concentration used in this study resulted in both good marking efficiency and low mortality of juveniles from the three cyprinid species. Before this study, daily growth increment formation had only been validated for eight cyprinid species. This study provides information on three additional species and further suggests that daily growth increments are reliable sources of age information for cyprinids. In addition, our results provide information for the conservation of two imperiled Brazos River fishes. Sharpnose and smalleye shiners were listed as candidate species for protection under the U.S. Endangered Species Act in 2002. The distribution and abundance of these two species have declined as a result of extensive modifications to the upper portion of the basin. As is the case for most small stream fishes, little biological data exist for members of the upper Brazos River fish assemblage. The threat of further water developments within the basin has precipitated efforts to gather much-needed life history information for members of the assemblage, including age and growth data. Validation of growth increment deposition is a necessary initial step to ensure the accuracy and utility of age and growth data that will be applied to the management and conservation of species in this assemblage. Because daily deposition of growth increments has been verified (this study), otolith microstructure can now be used to assess how
recruitment is associated with key environmental variables, such as stream discharge (e.g., Durham and Wilde 2005, 2006; Durham 2007). In addition, this information is beginning to provide insights into the connection between early life history events and overall population dynamics for these species (Durham 2007). Whenever possible, nonlethal methods of determining age are preferable to lethal methods, particularly when dealing with species of conservation concern. However, otoliths provide the only direct method of assessing daily age; consequently, critically important details, such as spawning and hatch dates, cannot be obtained with nonlethal methods of aging (e.g., scales and fins). The only nonlethal aging alternative that may be possible is the use of total length as a proxy of age. Regression models for total length and total number of growth increments in otoliths of sharpnose shiners (n ¼ 62; r2 ¼ 0.53; P , 0.0001), smalleye shiners (n ¼ 64; r2 ¼ 0.50; P , 0.0001), and plains minnow (n ¼ 31; r2 ¼ 0.26; P ¼ 0.0032) used in this study were significant, although length explained no more than 53% of the variation in total age. Durham and Wilde (2005) found that length was a good predictor of age for five cyprinid species in the Canadian River, Texas. In their study, length explained between 57% and 91% of variation in total number of daily growth increments. We advise caution when using this method because of the potential for environmental conditions to alter growth trajectories within a year (e.g., Durham and Wilde 2005). Acknowledgments We thank C. Hunt and R. Hunt for field assistance and C. Chizinski and D. Knabe for laboratory assistance and reading otoliths. The Texas Parks and Wildlife Department and U.S. Fish and Wildlife Service provided funding for this project. References Beamish, R. J., and G. A. McFarlane. 1983. The forgotten requirement for age validation in fisheries biology. Transactions of the American Fisheries Society 112: 735–743. Beckman, D. W., and R. G. Schulz. 1996. A simple method for marking fish otoliths with alizarin compounds. Transactions of the American Fisheries Society 125:146–149. Brouder, M. J. 2005. Age and growth of roundtail chub in the upper Verde River, Arizona. Transactions of the American Fisheries Society 134:866–871. Campana, S. E. 2001. Accuracy, precision, and quality control in age determination, including a review of the use and abuse of age validation methods. Journal of Fish Biology 59:197–242. Campana, S. E., and J. D. Neilson. 1985. Microstructure of
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