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Test and application of a non-destructive photo-method investigating the parasitic stage of the threatened mussel Margaritifera margaritifera on its host fish E. Salmo trutta
Biological Conservation 144 (2011) 2984–2990
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Test and application of a non-destructive photo-method investigating the parasitic stage of the threatened mussel Margaritifera margaritifera on its host fish Salmo trutta Martin E. Österling ⇑ Department of Biology, Karlstad University, SE 651 88 Karlstad, Sweden
a r t i c l e
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Article history: Received 27 May 2011 Received in revised form 31 August 2011 Accepted 11 September 2011 Available online 5 October 2011 Keywords: Density estimate Host-parasite Larval encystment Margaritifera margaritifera Photo-method PIT-tag Restoration Salmo trutta Threatened species Unionoida Ensystment intensity Glochidia larvae
a b s t r a c t The objective was to test the application of a novel, non-destructive photo-method estimating the larval encystment of one of the highly threatened unionid mussels, the freshwater pearl mussel (Margaritifera margaritifera) on the gills of its host fish, brown trout (Salmo trutta). There were significant correlations between the encystment intensity based on microscope counts and using the new photo-method for both young-of-the-year and older brown trout just after the encystment in October 2007 and just before larval release from the host fish in June 2008. The mean encystment intensity based on the two methods did not differ from each other for the two age classes of trout when based on comparisons including all individuals. An aquaria experiment showed that there were no differences in survival or growth between fish subjected to the treatments: photo-method and individual marking, photo-method and a control. When applied to encystment in single streams, there were significant correlations between the mean encystment intensity in each stream based on the methods for both trout age classes. Therefore, it may be possible to get reliable estimation of the encystment rates without injuring the mussel or the host fish, which may also be used in restoration and cultivation work. Furthermore, the larvae of M. margaritifera are among the smallest of all the worldwide-distributed, threatened unionid mussel species. The photomethod may therefore also be used for other mussel species with larger larvae, as they are more easily recognized on photos. Therefore, it may now be possible to investigate every life stage of unionid species without using harmful methods at all. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Reliable estimation of population density is fundamental in ecology, since it affects properties such as the estimation of population growth and status of populations (Stewart et al., 2001; Taylor and Pollard, 2008; Jones, 2011). Many methods used to estimate population density involve sampling methods that harm or even kill individuals. For threatened species, such destructive methods may cause substantial losses that are potential threats to these populations (Awruch et al., 2008; Rowcliffe et al., 2008; Österling et al., 2008). Still, estimates of abundance of threatened species are essential to be able to make the right conservation decisions (Rovero and Marshall, 2009; Tugores et al., 2010). The conservation of threatened species may include investigating which life stage limits a population. For threatened species with a parasitic life-stage on a host, this means that the parasitic life-stage also needs investigation (Bogan, 1993; Hastie and Young, 2001). In this case, destructive methods may not only harm the ⇑ Tel.: +46 54 700 18 02; fax: +46 700 14 62. E-mail address: [email protected] 0006-3207/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocon.2011.09.001
threatened species, but also its host (Österling et al., 2008). Functioning host populations are however needed to sustain the threatened species (Haag and Warren, 1998, 2003; Vaughn and Taylor, 2000). Therefore, the risk of reducing the parasite and the host during this life stage means that it is vital that sampling methods are non-destructive at the parasitic life stage. Mussels in the order unionoida, one of the most threatened groups in freshwater, are obligate temporary parasites on one or more fish species (Bogan, 1993; Haag and Warren, 1997; Strayer et al., 2004). The parasitic stage is therefore one of the life stages that potentially hinders recruitment of new mussels to the benthic population (Strayer et al., 2004; McNichols et al., 2011). Today, the method commonly used to investigate the parasitic stage is to kill the fish, cut out the gills and count the number of larvae using a microscope (Österling et al., 2008). However, low host fish densities and low encystment intensity on the host fish may make destructive sampling methods inappropriate, motivating the need to develop non-destructive methods to protect both the host fish population and the mussel parasite. The threatened freshwater pearl mussel (Margaritifera margaritifera) is a gill parasite on brown trout (Salmo trutta) or Atlantic
M.E. Österling / Biological Conservation 144 (2011) 2984–2990
salmon (Salmo salar) (Young and Williams, 1984; Hastie and Young, 2001). The mussels reproduce during summer, whereupon the mussels are gravid for about one month. When the glochidia larvae are ripe, which occur approximately between July and September, larvae are released from the gravid mussels (Hastie and Young, 2003). Upon release, the larvae must encounter and be encapsulated on the gills of a host fish. The larvae then live as parasites until they release from the fish, which occurs approximately between June and July the following year (Hastie and Young, 2001). Young-of-the-year (YOY) fish have been suggested to be important hosts, because they are often numerous and lack a well-developed immune response to the encystment of glochidia larvae (Bauer and Vogel, 1987; Bauer, 1987b). Older fish (one year or older) appear to have a stronger immune response, and the larvae may therefore fall off older fish before completing their parasitic life cycle stage (Bauer, 1987c). Therefore, reliable, non-harmful methods are needed to be developed for comparisons of encystments on YOY fish and older fish. This investigation tested (1) the reliability of photography as a novel, non-harmful method for measuring the encystment of larvae of M. margaritifera on the gills of its host fish, S. trutta, and (2) the application of the method on YOY and older brown trout populations separately, as the encystment capacity may differ depending on the age of the fish. Specifically, one aim was to investigate if it is possible to get reliable estimates of the encystment intensity just after the larval release in autumn, when the larvae are smallest. If so, the photo-method may be applicable to other mussel species, as the larvae of M. margaritifera are among the smallest of all unionoid larvae and therefore less easy to recognize compared to mussel species with larger larvae. Another aim was to see if it is possible to get reliable estimates of the potential number of mussels that will be released from the fish in early summer and recruit to the benthic population. The third aim was to test if the host fish suffered from the photo-method. Lastly, to test an application of the method, the mean encystment intensity based on the photo-method and microscope counts were compared in several streams.
2. Materials and methods The investigation was performed in the Ljungan catchment in central Sweden (68°99´–69°49´N, 14°65´–15°65´E). One stream (Hiån) was electrofished for brown trout in October 2007, which was approximately one month after the last gravid mussel was observed. Only one stream was electrofished since the trout densities are generally low in most of these streams and the local authorities wanted to avoid to disturbing these vulnerable populations. Eighteen streams were electrofished for brown trout in June 2008, which was about one week after the start of the temperature rise just after the spring flood, which is supposed to be the period when the parasitic mussel larvae have a few weeks left before they are released from the host fish (Hastie and Young, 2001). Electrofishing of brown trout was performed with a petrol-driven electroshocker (LUGAB). Thirty-eight trout individuals were caught in Hiån in October 2007, and 126 trout individuals were caught in the eighteen streams in June 2008. Captured trout were anesthetized with tricain methane-sulfonate (MS222), whereupon length and weight were measured, and a photograph was taken of the glochidia larvae on the gill filaments on the first gill arch. Photographs were taken by placing a flat, 5 mm wide steel spatula in the fish’s mouth so that it could be placed between the first and the second gill arch, thereby using the spatula as a background (Fig. 1). The steel spatula was always placed under the gill filaments in the middle of the first gill arch. The steel spatula was chosen as the larvae was best identified on the photograph with the
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Fig. 1. A photograph of encapsulated freshwater pearl mussel larvae on gill filaments on brown trout in spring 2008.
steel spatula as a background compared to other backgrounds, as white and black backgrounds, which was tested on trout on the lab prior to the field investigation. The photographs were taken with a Nikon Coolpix 995, through a Nikon SMZ800 stereo microscope. Each trout spent less than one minute out of the water when measured. The trout were then kept in buckets with fresh stream water that was exchanged every 5–10 min. There were no visual signs of injury, and just like a regular electro fishing event, every trout individual seemed as alert as before the measurements approximately forty-five minutes after the last fish had been measured (own observation). In this investigation, however, the fish were killed, and placed in individual, marked test tubes and preserved in 4% formalin. This was done to be able to compare the number of mussel larvae as determined from the photograph with the number of mussel larvae when counted on the dead fish under the microscope. In the laboratory, the gills were removed from the fish with a razor blade, and the number of larvae in the spatula area on the photo (the front side of the gill) was counted under a microscope. For comparisons with the photo-method, the number of larvae from the preserved gills were counted using the spatula area as done in the photo-method. The number of larvae on the gill on the other side of the fish (i.e. the backside of the gill, not photographed), was also counted under a microscope. Lastly, the total number of larvae on the preserved gills was counted under a microscope. The number of encapsulated larvae on the trout gills was estimated by constructing a formula based on the number of larvae at the spatula area on the photograph. The encystment intensity (i.e. the numbers of larvae per gill on each trout individual), Gtrout, was calculated by the formula:
Gtrout ¼ ðGphoto K Q =MÞ Gmean all gill arches =Gmean gill arch 1 : Gphoto is the number of larvae counted at the spatula area on the photo. K is the number of larvae counted at the front side and the back side of the gill at the spatula area, divided by the number of larvae counted at the front side of the gill at the spatula area (i.e. a factor relating the total number of larvae on the spatula area to the number of larvae at the spatula area on the photograph). K was 2.0 for YOY trout and 1.8 for older trout. Q is a factor correcting for the difference in larval density between the spatula area and the area outside the spatula area. Q was added since M (below) assumed uniform larval density over the entire 1st gill. Therefore, Q = A + BC, where A is the mean proportion of the gill area covered by the spatula area, B is the mean proportion of the gill area outside
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Table 1 Correlation between encystment intensity based on microscope counts and the photo-method in Hiån in October 2007. Methods used in the estimation of the proportion of the number of larvae at the spatula area on the 1st gill arch (M). Mean numbers of larvae on YOY and older trout in Hiån 2007 based on the methods estimating P. Correlations between encystment intensity (Gtrout) based on microscope counts and the photo-method. YOY trout (n = 20) and older trout (n = 13) YOY
Method used in the estimation of the proportion of numbers of larvae at the spatula area on the 1st gill arch (M), r2, p
Mean encystment intensity (Gtrout) on YOY in Hiån, 2007 (larvae gill1, n = 20)
Mean encystment intensity (Gtrout) on older trout in Hiån, 2007 (larvae gill1, n = 13)
Microscope counts 0.0029 L + 0.58 (regression), 0.85, <0.05 26.8 L1.0 (Exp., base 10), 0.89, <0.05 0.78 e0.01L (Exp., base e), 0.89, <0.05 0.28 Ln(L) + 1.58 (natural log), 0.86, <0.05 0.01 W + 0.41 (regression), 0.82, <0.05 0.55 W0.34 (Exp, base 10), 0.88, <0.05 0.42 e0.037W (Exp, base e), 0.87, <0.05 0.095 Ln (W) + 0.49 (nat. log), 0.85, <0.05 Gmean photo/Gmean gill arch 1 (Mean prop of larvae on photo)
26.3 ± 5.8 24.4 ± 6.4 25.1 ± 6.7 24.8 ± 6.6 24.6 ± 6.5 24.3 ± 6.4 25.2 ± 6.8 24.6 ± 6.5 24.8 ± 6.6 29.7 ± 7.7
72.0 ± 27.1 77.5 ± 28.0 73.6 ± 26.3 74.0 ± 26.5 73.3 ± 26.3 73.4 ± 26.8 74.0 ± 26.4 75.1 ± 27.2 73.9 ± 26.5 73.9 ± 26.5
Older trout
r
p
r
p
– 0.95 0.94 0.94 0.94 0.95 0.94 0.95 0.94 0.95
– <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
– 0.98 0.98 0.98 0.98 0.99 0.98 0.99 0.98 0.97
– <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
of the spatula area, and C is the proportion of larvae per gill area for the gill region outside of the spatula area. This resulted in Q = 0.77 for YOY trout and Q = 0.76 for older trout from Hiån 2007. In 2008, Q = 0.87 for YOY trout, and Q = 0.89 for older trout. M is the estimate of the proportion of the spatula area on the photo in relation to the total area of the 1st gill arch for each trout. To calculate M, weight and length of the fish were related to the proportions of the numbers of gill filaments in the spatula area. Four equations with length and four with weight as the independent variables were tested based on linear regression, natural logarithm, exponential equation with base 10, and exponential equation with base e. This resulted in eight equations, which were all significant (M in the formula; Table 1). To obtain mean larval densities per gill, the number of larvae on the first gill arch also needed to be related to the mean number of larvae per gill arch. This was done by dividing Gmean all gill arches, which is the mean number of larvae on all gill arches, and Gmean gill arch 1, which is the mean number of larvae on the first gill arch. Lastly, a ninth equation used the proportion between the mean number of larvae at the spatula area on the photo, Gmean photo, and the mean number of larvae per gill, Gmean gill arch 1 instead of R/P. In this case, Gtrout, was calculated by the formula: Gtrout ¼ ðGphoto K Gmean photo =Gmean gill arch 1 Þ Gmean all gill arches =Gmean gill arch 1 :
The effect of subjecting the fish to the photograph method on trout survival and growth was tested in an aquaria experiment. Hatchery reared YOY trout from the Gullspång River were used in this experiment. Three different treatments, using 10 trout individuals per treatment, were compared. In the first treatment, the fish were tagged with PIT tags inserted into the peritoneal cavity, were measured for length and weight, and subjected to a simulated photo session by placing the spatula between the first and the second gill arch for one minute. This length of time was not exceeded in the field, and thus considered the maximum stress any individual fish experienced out of the water. The second treatment included PIT-tagging (Trovan microtransponder 101, 11.5 mm) and measures of length and weight. The third treatment was a control where only length and weight were measured. All trout were anesthetized with tricain methane-sulfonate (MS222) before the treatments were performed. The PIT-tag insertion treatment was tested as this is often used to mark trout in experiments, and was expected to increase the stress experienced the trout as compared to a treatment without PIT-tagging, as in the field investigation. The aquaria experiment was performed from 2008-11-10 to 2008-12-23 in 100 L aquaria with EHEIM 2217 pumps and standard filters. Temperature was measured every hour with a
HOBO Pendant Temperature/Light Data Logger. The mean water temperature was 11.2 ± 0.5 (SD) °C over the experimental period. Approximately half of the water in the aquaria was exchanged once a week. The trout were fed 4% of their body weight five times a week with defrosted chironomid larvae. Weight and length measures were performed every 2nd week on trout after anesthetizing with tricain methane-sulfonate (MS222). The relationship between the number of larvae in the spatula area on the photo and the actual number of larvae in the spatula area counted under microscope, and the relationship between the number of larvae per gill based on the photo-method and the numbers of larvae based on counting the larvae under microscope were analyzed using correlations. The relationship between the mean number of larvae per gill in each stream based on the photo-method and the numbers of larvae based on counting the larvae under microscope was also analyzed using correlations. The differences in mean larval encystment intensity in the streams based on the photo-method and mean larval encystment intensity based on counting the larvae under microscope were compared using paired t-tests. The differences in length, weight, length change and weight change in the aquaria experiment were analyzed using ANOVA:s. All measures of variation in the results section are given as ±1 SE. 3. Results There were significant correlations between the number of larvae in the spatula area counted on the preserved gills under the microscope and the number of larvae at the spatula area counted on the photograph on the trout from Hiån in October 2007 (Fig. 2a) and on every trout from all streams in June 2008 (Fig. 2b). 3.1. Encystment on individual brown trout in spring 2007 All correlations between encystment intensity based on the counted numbers of larvae under the microscope and encystment intensity estimated by using the photo-method for YOY and older in 2007 were significant (Table 1). Significant correlations with the highest r-values are shown for YOY and for older trout (Fig. 3). The mean encystment intensity was 26.3 ± 5.8 larvae gill1 and 72.0 ± 27.1 larvae gill1 on the YOY and older trout from Hiån in October 2007 respectively, based on the counted numbers of larvae under the microscope. These encystment rates did not differ significantly from the mean encystment of larvae based on the photo-method from Hiån in October 2007, which were between
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The mean difference and the absolute difference between encystment intensity based on the microscope counts and the photo-method in 2007 was 2.0 ± 2.3 larvae gill1 and 6.9 ± 1.7 larvae gill1 for YOY trout, and 1.4 ± 4.2 larvae gill1 and 10.4 ± 2.9 larvae gill1 for older trout. Seven of the 20 YOY trout individuals and four of the thirteen older trout individuals from 2007 lacked larvae on the spatula area, but were found to have low encystments intensities when counted under the microscope. The mean encystment intensity of these ‘‘zeros’’ was 1.5 ± 0.9 larvae gill1 on YOY trout and 9.6 ± 5.6 larvae gill1 and on older trout in 2007 when counted under the microscope. 3.2. Encystment on individual brown trout in spring 2008 Fig. 2a. Correlation between encystment intensity at the spatula area counted in microscope and encystment intensity at the spatula area counted on the photo on trout from Hiån autumn 2007 (r = 0.95, p < 0.0001, n = 25).
Fig. 2b. Correlation between encystment intensity at the spatula area counted in microscope and encystment intensity at the spatula area counted on the photo on trout from all streams spring 2008 (r = 0.99, p < 0.0001, n = 126).
There were significant correlations between encystment intensity based on microscope counts and the photo-method for both YOY and older trout in 2008 (Table 2). The correlation between the estimates of the encystment intensity based on the photomethod and the microscope counts had the highest r-values when using the estimation of P based on the weight and exponential equation for YOY trout, and P based on length and regression equation for older trout (Fig. 4). The mean encystment intensity on 0+ trout was 36.4 ± 6.1 larvae gill1 in 2008 based on microscope counts, and did not differ significantly from the photo-method, which was between 35.2 ± 5.9 and 39.0 ± 6.6 larvae gill1 (paired t-tests, p > 0.05). Gtrout based on the natural logarithm length equation (36.6 ± 6.2 larvae gill1) was closest to the mean encystment intensity based on microscope counts for YOY trout. The mean encystment intensity on older trout was 12.9 ± 4.2 larvae gill1 in 2008 based on microscope counts, and did not differ from the mean encystment intensity when based on the photo-method, which was between 12.2 ± 4.0 and 15.0 ± 4.9 larvae gill1 (paired t-tests, p > 0.05). Gtrout based on Gmean photo/Gmean gill arch 1 (12.2 ± 4.0 larvae gill1) was closest to the Gtrout based on microscope counts (Table 2). The mean difference and the absolute difference between encystment intensity based on microscope counts and the photomethod was 2.5 ± 1.4 larvae trout1 and 6.7 ± 1.1 larvae trout1 for YOY trout, and 0.72 ± 1.2 larvae trout1 and 4.4 ± 1.0 larvae trout1 for older trout in 2008 respectively. Six of the 63 YOY trout individuals and 12 of the 64 older trout individuals from 2008 that lacked larvae on the photo were found to have larvae when counted under the microscope. The mean encystment intensity of these ‘‘zeros’’ was 2.1 ± 1.3 larvae gill1 and 2.7 ± 1.0 on YOY and older trout respectively in 2008 when counted under the microscope. 3.3. Survival and growth of brown trout in aquaria experiment
Fig. 3. Correlations between the encystment intensity based on counting the larvae in microscope and based on the photo-method for YOY trout (squares; r = 0.95, p < 0.0001, n = 25) and for older trout (triangles; r = 0.99, p < 0.0001, n = 13) in 2007. The estimation of P for YOY and older trout is based on the proportion of filaments on the spatula area at the photo, and the exponential equation with the base e and weight, respectively.
24.3 ± 6.4 larvae gill1 and 29.7 ± 7.7 larvae gill1 on YOY trout and between 73.3 ± 26.3 larvae gill1 and 77.5 ± 28.0 larvae gill1 on older trout (paired t-tests, p > 0.05). The mean encystment intensity of larvae based on the formula using the estimation of P based on length and exponential equation with the base 10 (25.1 ± 6.7 larvae gill1) and based on length and natural logarithm equation (73.3 ± 26.3 larvae gill1) were closest to mean encystment of larvae for YOY trout and for older trout respectively based on counted number of larvae under the microscope (Table 1).
Twenty-five out of 30 trout individuals survived the 30 days aquarium experiment. Eight trout individuals survived the treatment with a PIT-tag, length and weight measure, and the simulated photo-method. Nine trout individuals survived the treatment with a PIT-tag and length and weight measure, while eight trout individuals survived the control treatment with only length and weight measure. There was no difference in weight or length of the trout between the three treatments at any of the four occasions when the trout was measured (ANOVAs, p > 0.05). Likewise, there was no difference in weight or length change between the three treatments after 10, 20 and 30 days (ANOVAs, p > 0.05, n = 25) (Fig. 5). 3.4. Mean encystment intensity in the streams The mean encystment intensity varied between 0 and 119 ± 41 larvae gill1 and 0 and 105 ± 38–122 ± 46 larvae gill1 on YOY trout (n = 1–18) and between 0 and 132 (one ind.) larvae gill1 and 0 and
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Table 2 Methods used in the estimation of the proportion of the number of larvae at the spatula area on the 1st gill arch (M). Mean larval encystment of YOY in all streams 2008. Correlation between encystment intensity based on microscope counts and the photo-method in Hiån in October 2007. Mean larval encystment of older trout in all streams 2008. Method used in the estimation of the proportion of numbers of larvae at the spatula area on the 1st gill arch (M), r2, p
Microscope counts 0.0029 L + 0.58 (regression), 0.85, <0.05 26.8 L1.0 (Exp., base 10), 0.89, <0.05 0.78 e0.01L (Exp., base e), 0.89, <0.05 0.28 Ln(L) + 1.58 (nat. log), 0.86, <0.05 0.01 W + 0.41 (regression), 0.82, <0.05 0.55 W0.34 (Exp, base 10), 0.88, <0.05 0.42 e0.037W (Exp, base e), 0.87, <0.05 0.095 Ln (W) + 0.49 (nat. log), 0.85, <0.05 Gmean photo/Gmean gill arch 1 (Mean prop of larvae on photo)
Mean encystment intensity of YOY in all streams 2008, (larvae gill1, n = 62)
36.4 ± 6.1 35.2 ± 5.9 37.5 ± 6.4 36.9 ± 6.2 36.6 ± 6.2 35.9 ± 5.9 39.0 ± 6.6 36.6 ± 6.1 37.8 ± 6.4 36.9 ± 6.1
Fig. 4. Correlations between encystment intensity based on counting the number of larvae under the microscope and based on the photo-method for YOY trout (squares; r = 0.98, p < 0.0001, n = 62) and for older trout (triangles; r = 0.97, p < 0.0001, n = 64) in 2008. The estimation of P for YOY and older trout is based on the exponential weight equation with base 10, and length and regression, respectively.
Correlations of the number of larvae per gill on YOY trout based on microscope count and the photo-method (Gtrout) in all streams in May 2008 (n = 62) r
p
– 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.97
– <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Mean encystment intensity of older trout in all streams 2008, (larvae gill1, n = 64)
12.9 ± 4.2 13.7 ± 4.5 14.6 ± 4.8 14.5 ± 4.8 14.4 ± 4.7 14.7 ± 4.9 15.0 ± 4.9 14.6 ± 4.8 14.9 ± 4.8 12.2 ± 4.0
Correlations between encystment intensity on older trout based on microscope counts and the photo-method (Gtrout) in all streams in May 2008 (n = 64) r
p
– 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.96
– <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Fig. 5. Weight of brown trout at the beginning of the aquaria experiment (0 days), after 10, 20 and 30 days in the treatments (1) control (triangles), (2) pit-tag marked fish (squares), (3) pit-tag marked fish and photo-method (diamonds).
4. Discussion 136–160 larvae gill1 on older trout (n = 1–10) in the 16 streams in 2008 when based on the microscope counts and the photo-method, respectively. The mean encystment intensity in the streams were estimated to be zero based on the photo-method when there in fact were mean encystments of 0.9 ± 0 (one individual), 1.0 ± 0 (one individual) and 2.7 ± 3.3 (one of three individuals infected) larvae gill1 on YOY trout, and 0.1 ± 0.07 (one of four individuals infected) and 0.5 ± 0.3 (two of three individuals infected) larvae gill1 on older trout. In the other streams, there were no differences in the mean encystment per gill when based on microscope counts versus the calculations based on the photo-method (paired t-tests, p > 0.05). There were significant correlations between the mean encystment intensity in each stream based on microscope counts and the photo-method for YOY and older brown trout in June 2008. Encystment intensity based on the photo-method had the highest r-values when P was based on the exponential length equation with base 10 for YOY trout, and on Gmean photo/Gmean gill arch 1 for older trout, respectively (Fig. 6).
This investigation showed that a non-destructive method could be used instead of a destructive method when investigating the parasitic stage of a unionid mussel species on its host fish. The photo-method used here measured the encystment of M. margaritifera glochidia larvae on S. trutta gills without injuring the fish, or causing reduced growth of the fish. Therefore, it may be possible to investigate every life-stage of the highly threatened unionoid mussels such as M. margaritifera using non-destructive methods (Hastie and Boon, 2001; Hastie and Young, 2003; Österling et al., 2008, 2010). All equations relating the number of larval encystments at the spatula area to the number of larval encystments at the complete area of the 1st gill arch could be used for the estimation of the numbers of larvae on each trout individual. This resulted in significant correlations between the estimation of encystment intensity based on the photo-method and the microscope counts. Consequently, each one of the equations relating the number of larval encystments at the spatula area to the number of larval encystments at the complete area of the 1st gill arch could be used when
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Fig. 6. Correlation between mean encystment intensity per stream based on counting the number of larvae under the microscope and the mean encystment intensity per stream based on the photo-method on YOY trout (squares; r = 0.99, p < 0.0001, n = 13) and for older trout (triangles; r = 0.99, p < 0.0001, n = 16) in 2008. The estimation of P for YOY and older trout is based on the exponential equation with the base 10 and length, and proportion of filaments on the spatula area at the photo, respectively.
estimating the number of larvae on M. margaritifera using the photo-method. However, the usefulness of these equations may differ between M. margaritifera distribution areas. Therefore, the equations could be tested in M. margaritifera populations in other parts of its distribution, and on other unionid mussel species using salmonids as hosts, as the morphology of their gills resemble brown trout gills. On other mussel species, using other fish species as hosts, the likelihood of host-species-specific adjustments of the photo-method probably increase. The estimation of the encystment intensity on individual fish using the photo-method was significantly related to the encystment intensity on individual fish based on microscope counts. In fact, the correlations explained ninety-four percent or more of the variation in the analyses, and the slopes of the correlations were close to one. Only occasionally, the encystment intensity differed as much as about two times when comparing the photomethod with the method based on counting the larvae under the microscope. However, the estimates of the two methods were always in the same order of magnitude. The photo-method showed a high precision when estimating the mean encystment intensity per stream on both YOY and older trout, which is often needed when investigating population regulation and host age-dependent sensitivity of this life-stage (Young and Williams, 1983, 1984; Bauer, 1987b, 1987c; Österling et al., 2008). Therefore, the photo-method may be applied when estimating mean encystment intensity in different mussel – brown trout streams. In fact, the photo-method seemed to be reliable even when there were small sample sizes (i.e. low brown trout density), which was the case for several of the individual streams. However, the estimation of the mean encystment intensity based on the photo-method was zero in a few occasions when there in fact were a few encystments based on counting the larvae under the microscope. This was mainly a result of few encystments on few trout individuals in streams with low mussel density which was missed on the relatively small area covered by the photographs. The mean encystment intensity in these measurements was only between 0.1 and 2.7 larvae gill1, which is a very low contribution to the population (Österling et al., 2008). However, the estimated encystment intensity based on the photo-method was never zero when there were at least five trout individuals. Often there is a possibility to catch five or more trout individuals when electro-fishing in M. margaritifera streams (Hastie and Boon, 2001; Hastie and Young, 2001; Geist et al., 2006; Österling et al., 2008). Therefore, it should be possible to capture enough trout individuals to make appropriate measurements of mean encystment intensity.
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The estimation of encystment intensity was slightly better in spring than in the autumn, probably because the larger larvae in the spring (Hastie and Young, 2001, 2003) were more easily recognized on the photo compared to the smaller larvae in the autumn. However, the strength of these estimations was generally high both in the autumn and in spring. Thus, it may also be possible to estimate changes in the encystment intensity between mussel and trout populations, and differences between different year classes of brown trout without harming the fish. In fact, it may even be possible to measure changes in encystment intensity on the individual fish, as the survival rates were high when marked with PIT-tags. Lastly, it would also be possible to establish relationships between the numbers of encysted larvae present in relation to the density of the mussel population within the stream. There are a number of applications in which this photo-method may be used when managing threatened unionoid mussel populations (Geist, 2010), apart for measuring mean encystment intensity. First, the photo-method may be used to select fish with high numbers of infested larvae. This may be needed at streams where cultivation measures are performed, as fish density may be a limiting factor both in streams and at breeding facilities. Fish with high larval load may therefore be selected to raise the numbers of juvenile mussels (Österling et al., 2008). Secondly, the photo-method combined with PIT-tagging did not seem to have any negative effects on growth or survival of the trout. Therefore, combining the photo-method with PIT-tagging may allow studies of changes in encystment intensity on individual fish. It would also be possible to study colonization distances of the mussel, if trout are marked and photographed at both capture and recapture events. Lastly, the photo-method could be tested for other mussel species, as the larvae of M. margaritifera are among the smallest of all unionid mussels and therefore less easy to detect compared to mussel species with larger glochidia larvae (Bauer, 1987a, 1998). If the photo-method is applicable to other unionid mussel species, it should be possible to investigate where in the life-cycle several unionid species are threatened without using harmful methods at all, as the juvenile and adult stages can already be investigated using non-harmful methods (Hastie and Boon, 2001; Young et al., 2001; Hastie and Young, 2003; Österling et al., 2008, 2010). Acknowledgments I thank Fortums Nordiska Miljöfond for financing this study, H. Söderberg for inspiration, H. Karlsson for field assistance, and B. Arvidsson, L. Greenberg and four anonymous referees for commenting earlier versions of the manuscript. The electro-fishing and all other fish handling was performed under licenses from the Country Administration in Västernorrland and from the Swedish Animal Welfare Agency CFN, Gothenburg (Dnr: A81-07). References Awruch, C.A., Frusher, S.D., Pankhurst, N.W., Stevens, J.D., 2008. Non-lethal assessment of reproductive characteristics for management and conservation of sharks. Marine Ecology – Progress Series 355, 277–285. Bauer, G., 1987a. Reproductive strategy of the freshwater pearl mussel Margaritifera margaritifera. Journal of Animal Ecology 56, 691–704. Bauer, G., 1987b. The parasitic stage of the freshwater pearl mussel (Margaritifera margaritifera L.) 2. Susceptibility of brown trout. Archiv Fur Hydrobiologie 76, 403–412. Bauer, G., 1987c. The parasitic stage of the freshwater pearl mussel (Margaritifera margaritifera L.) 3. Host relationships. Archiv Fur Hydrobiologie 76, 413–423. Bauer, G., 1998. Allocation policy of female freshwater pearl mussels. Oecologia 117, 90–94. Bauer, G., Vogel, C., 1987. The parasitic stage of the freshwater pearl mussel (Margaritifera margaritifera L.) 1. Host response to glochidiosis. Archiv Fur Hydrobiologie 76, 393–402. Bogan, A.E., 1993. Fresh-water bivalve extinctions (Mollusca, Unionoida) – a search for causes. American Zoologist 33, 599–609.
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Geist, J., 2010. Strategies for the conservation of endangered freshwater pearl mussels (Margaritifera margaritifera L.): a synthesis of Conservation Genetics and Ecology. Hydrobiologia 644, 69–88. Geist, J., Porkka, M., Kuehn, R., 2006. The status of host fish populations and fish species richness in European freshwater pearl mussel (Margaritifera margaritifera) streams. Aquatic Conservation – Marine and Freshwater Ecosystems 16, 251–266. Haag, W.R., Warren, M.L., 1997. Host fishes and reproductive biology of 6 freshwater mussel species from the Mobile Basin, USA. Journal of the North American Benthological Society 16, 576–585. Haag, W.R., Warren, M.L., 1998. Role of ecological factors and reproductive strategies in structuring freshwater mussel communities. Canadian Journal of Fisheries and Aquatic Sciences 55, 297–306. Haag, W.R., Warren, M.L., 2003. Host fishes and encystment strategies of freshwater mussels in large Mobile Basin streams, USA. Journal of the North American Benthological Society 22, 78–91. Hastie, L.C., Boon, P.J., 2001. Does electrofishing harm freshwater pearl mussels? Aquatic Conservation – Marine and Freshwater Ecosystems 11, 149–152. Hastie, L.C., Young, M.R., 2001. Freshwater pearl mussel (Margaritifera margaritifera) glochidiosis in wild and farmed Salmonid stocks in Scotland. Hydrobiologia 445, 109–119. Hastie, L.C., Young, M.R., 2003. Timing of spawning and glochidial release in Scottish freshwater pearl mussel (Margaritifera margaritifera) populations. Freshwater Biology 48, 2107–2117. Jones, J.P.G., 2011. Monitoring species abundance and distribution at the landscape scale. Journal of Applied Ecology 48, 9–13. McNichols, K.A., Mackie, G.L., Ackerman, J.D., 2011. Host fish quality may explain the status of endangered Epioblasma torulosa rangiana and Lampsilis fasciola (Bivalvia: Unionidae) in Canada. Journal of the North American Benthological Society 30, 60–70. Österling, E.M., Greenberg, L.A., Arvidsson, B.L., 2008. Relationship of biotic and abiotic factors to recruitment patterns in Margaritifera margaritifera. Biological Conservation 141, 1365–1370.
Österling, M.E., Arvidsson, B.L., Greenberg, L.A., 2010. Habitat degradation and the decline of the threatened mussel Margaritifera margaritifera: influence of turbidity and sedimentation on the mussel and its host. Journal of Applied Ecology 47, 759–768. Rovero, F., Marshall, A.R., 2009. Camera trapping photographic rate as an index of density in forest ungulates. Journal of Applied Ecology 46, 1011–1017. Rowcliffe, J.M., Field, J., Turvey, S.T., Carbone, C., 2008. Estimating animal density using camera traps without the need for individual recognition. Journal of Applied Ecology 45, 1228–1236. Stewart, K.E.J., Bourn, N.A.D., Thomas, J.A., 2001. An evaluation of three quick methods commonly used to assess sward height in ecology. Journal of Applied Ecology 38, 1148–1154. Strayer, D.L., Downing, J.A., Haag, W.R., King, T.L., Layzer, J.B., Newton, T.J., Nichols, S.J., 2004. Changing perspectives on pearly mussels, North America’s most imperiled animals. Bioscience 54, 429–439. Taylor, S.L., Pollard, K.S., 2008. Evaluation of two methods to estimate and monitor bird populations. Plos One 3, e3047. Tugores, M.P., Iglesias, M., Diaz, N., Onate, D., Miquel, J., Giraldez, A., 2010. Latitudinal and interannual distribution of the European anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) in the western Mediterranean, and sampling uncertainty in abundance estimates. ICES Journal of Marine Sciences 67, 1574–1586. Vaughn, C.C., Taylor, C.M., 2000. Macroecology of a host-parasite relationship. Ecography 23, 11–20. Young, M., Williams, J., 1983. The reproductive biology of the freshwater pearl mussel Margaritifera margaritifera (Linn.) in Scotland 2. Laboratory studies. Archiv Fur Hydrobiologie 100, 29–43. Young, M., Williams, J., 1984. The reproductive biology of the freshwater pearl mussel Margaritifera margaritifera (Linn.) in Scotland 1. Field studies. Archiv Fur Hydrobiologie 99, 405–422. Young, M.R., Cosgrove, P.J., Hastie, L.C., Henniger, B., 2001. A standardised method for assessing the status of freshwater mussels in clear, shallow rivers. Journal of Molluscan Studies 67, 395–396.
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Test and application of a non-destructive photo-method investigating the parasitic stage of the threatened mussel Margaritifera margaritifera on its host fish Salmo trutta Martin E. Österling ⇑ Department of Biology, Karlstad University, SE 651 88 Karlstad, Sweden
a r t i c l e
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Article history: Received 27 May 2011 Received in revised form 31 August 2011 Accepted 11 September 2011 Available online 5 October 2011 Keywords: Density estimate Host-parasite Larval encystment Margaritifera margaritifera Photo-method PIT-tag Restoration Salmo trutta Threatened species Unionoida Ensystment intensity Glochidia larvae
a b s t r a c t The objective was to test the application of a novel, non-destructive photo-method estimating the larval encystment of one of the highly threatened unionid mussels, the freshwater pearl mussel (Margaritifera margaritifera) on the gills of its host fish, brown trout (Salmo trutta). There were significant correlations between the encystment intensity based on microscope counts and using the new photo-method for both young-of-the-year and older brown trout just after the encystment in October 2007 and just before larval release from the host fish in June 2008. The mean encystment intensity based on the two methods did not differ from each other for the two age classes of trout when based on comparisons including all individuals. An aquaria experiment showed that there were no differences in survival or growth between fish subjected to the treatments: photo-method and individual marking, photo-method and a control. When applied to encystment in single streams, there were significant correlations between the mean encystment intensity in each stream based on the methods for both trout age classes. Therefore, it may be possible to get reliable estimation of the encystment rates without injuring the mussel or the host fish, which may also be used in restoration and cultivation work. Furthermore, the larvae of M. margaritifera are among the smallest of all the worldwide-distributed, threatened unionid mussel species. The photomethod may therefore also be used for other mussel species with larger larvae, as they are more easily recognized on photos. Therefore, it may now be possible to investigate every life stage of unionid species without using harmful methods at all. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Reliable estimation of population density is fundamental in ecology, since it affects properties such as the estimation of population growth and status of populations (Stewart et al., 2001; Taylor and Pollard, 2008; Jones, 2011). Many methods used to estimate population density involve sampling methods that harm or even kill individuals. For threatened species, such destructive methods may cause substantial losses that are potential threats to these populations (Awruch et al., 2008; Rowcliffe et al., 2008; Österling et al., 2008). Still, estimates of abundance of threatened species are essential to be able to make the right conservation decisions (Rovero and Marshall, 2009; Tugores et al., 2010). The conservation of threatened species may include investigating which life stage limits a population. For threatened species with a parasitic life-stage on a host, this means that the parasitic life-stage also needs investigation (Bogan, 1993; Hastie and Young, 2001). In this case, destructive methods may not only harm the ⇑ Tel.: +46 54 700 18 02; fax: +46 700 14 62. E-mail address: [email protected] 0006-3207/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocon.2011.09.001
threatened species, but also its host (Österling et al., 2008). Functioning host populations are however needed to sustain the threatened species (Haag and Warren, 1998, 2003; Vaughn and Taylor, 2000). Therefore, the risk of reducing the parasite and the host during this life stage means that it is vital that sampling methods are non-destructive at the parasitic life stage. Mussels in the order unionoida, one of the most threatened groups in freshwater, are obligate temporary parasites on one or more fish species (Bogan, 1993; Haag and Warren, 1997; Strayer et al., 2004). The parasitic stage is therefore one of the life stages that potentially hinders recruitment of new mussels to the benthic population (Strayer et al., 2004; McNichols et al., 2011). Today, the method commonly used to investigate the parasitic stage is to kill the fish, cut out the gills and count the number of larvae using a microscope (Österling et al., 2008). However, low host fish densities and low encystment intensity on the host fish may make destructive sampling methods inappropriate, motivating the need to develop non-destructive methods to protect both the host fish population and the mussel parasite. The threatened freshwater pearl mussel (Margaritifera margaritifera) is a gill parasite on brown trout (Salmo trutta) or Atlantic
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salmon (Salmo salar) (Young and Williams, 1984; Hastie and Young, 2001). The mussels reproduce during summer, whereupon the mussels are gravid for about one month. When the glochidia larvae are ripe, which occur approximately between July and September, larvae are released from the gravid mussels (Hastie and Young, 2003). Upon release, the larvae must encounter and be encapsulated on the gills of a host fish. The larvae then live as parasites until they release from the fish, which occurs approximately between June and July the following year (Hastie and Young, 2001). Young-of-the-year (YOY) fish have been suggested to be important hosts, because they are often numerous and lack a well-developed immune response to the encystment of glochidia larvae (Bauer and Vogel, 1987; Bauer, 1987b). Older fish (one year or older) appear to have a stronger immune response, and the larvae may therefore fall off older fish before completing their parasitic life cycle stage (Bauer, 1987c). Therefore, reliable, non-harmful methods are needed to be developed for comparisons of encystments on YOY fish and older fish. This investigation tested (1) the reliability of photography as a novel, non-harmful method for measuring the encystment of larvae of M. margaritifera on the gills of its host fish, S. trutta, and (2) the application of the method on YOY and older brown trout populations separately, as the encystment capacity may differ depending on the age of the fish. Specifically, one aim was to investigate if it is possible to get reliable estimates of the encystment intensity just after the larval release in autumn, when the larvae are smallest. If so, the photo-method may be applicable to other mussel species, as the larvae of M. margaritifera are among the smallest of all unionoid larvae and therefore less easy to recognize compared to mussel species with larger larvae. Another aim was to see if it is possible to get reliable estimates of the potential number of mussels that will be released from the fish in early summer and recruit to the benthic population. The third aim was to test if the host fish suffered from the photo-method. Lastly, to test an application of the method, the mean encystment intensity based on the photo-method and microscope counts were compared in several streams.
2. Materials and methods The investigation was performed in the Ljungan catchment in central Sweden (68°99´–69°49´N, 14°65´–15°65´E). One stream (Hiån) was electrofished for brown trout in October 2007, which was approximately one month after the last gravid mussel was observed. Only one stream was electrofished since the trout densities are generally low in most of these streams and the local authorities wanted to avoid to disturbing these vulnerable populations. Eighteen streams were electrofished for brown trout in June 2008, which was about one week after the start of the temperature rise just after the spring flood, which is supposed to be the period when the parasitic mussel larvae have a few weeks left before they are released from the host fish (Hastie and Young, 2001). Electrofishing of brown trout was performed with a petrol-driven electroshocker (LUGAB). Thirty-eight trout individuals were caught in Hiån in October 2007, and 126 trout individuals were caught in the eighteen streams in June 2008. Captured trout were anesthetized with tricain methane-sulfonate (MS222), whereupon length and weight were measured, and a photograph was taken of the glochidia larvae on the gill filaments on the first gill arch. Photographs were taken by placing a flat, 5 mm wide steel spatula in the fish’s mouth so that it could be placed between the first and the second gill arch, thereby using the spatula as a background (Fig. 1). The steel spatula was always placed under the gill filaments in the middle of the first gill arch. The steel spatula was chosen as the larvae was best identified on the photograph with the
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Fig. 1. A photograph of encapsulated freshwater pearl mussel larvae on gill filaments on brown trout in spring 2008.
steel spatula as a background compared to other backgrounds, as white and black backgrounds, which was tested on trout on the lab prior to the field investigation. The photographs were taken with a Nikon Coolpix 995, through a Nikon SMZ800 stereo microscope. Each trout spent less than one minute out of the water when measured. The trout were then kept in buckets with fresh stream water that was exchanged every 5–10 min. There were no visual signs of injury, and just like a regular electro fishing event, every trout individual seemed as alert as before the measurements approximately forty-five minutes after the last fish had been measured (own observation). In this investigation, however, the fish were killed, and placed in individual, marked test tubes and preserved in 4% formalin. This was done to be able to compare the number of mussel larvae as determined from the photograph with the number of mussel larvae when counted on the dead fish under the microscope. In the laboratory, the gills were removed from the fish with a razor blade, and the number of larvae in the spatula area on the photo (the front side of the gill) was counted under a microscope. For comparisons with the photo-method, the number of larvae from the preserved gills were counted using the spatula area as done in the photo-method. The number of larvae on the gill on the other side of the fish (i.e. the backside of the gill, not photographed), was also counted under a microscope. Lastly, the total number of larvae on the preserved gills was counted under a microscope. The number of encapsulated larvae on the trout gills was estimated by constructing a formula based on the number of larvae at the spatula area on the photograph. The encystment intensity (i.e. the numbers of larvae per gill on each trout individual), Gtrout, was calculated by the formula:
Gtrout ¼ ðGphoto K Q =MÞ Gmean all gill arches =Gmean gill arch 1 : Gphoto is the number of larvae counted at the spatula area on the photo. K is the number of larvae counted at the front side and the back side of the gill at the spatula area, divided by the number of larvae counted at the front side of the gill at the spatula area (i.e. a factor relating the total number of larvae on the spatula area to the number of larvae at the spatula area on the photograph). K was 2.0 for YOY trout and 1.8 for older trout. Q is a factor correcting for the difference in larval density between the spatula area and the area outside the spatula area. Q was added since M (below) assumed uniform larval density over the entire 1st gill. Therefore, Q = A + BC, where A is the mean proportion of the gill area covered by the spatula area, B is the mean proportion of the gill area outside
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Table 1 Correlation between encystment intensity based on microscope counts and the photo-method in Hiån in October 2007. Methods used in the estimation of the proportion of the number of larvae at the spatula area on the 1st gill arch (M). Mean numbers of larvae on YOY and older trout in Hiån 2007 based on the methods estimating P. Correlations between encystment intensity (Gtrout) based on microscope counts and the photo-method. YOY trout (n = 20) and older trout (n = 13) YOY
Method used in the estimation of the proportion of numbers of larvae at the spatula area on the 1st gill arch (M), r2, p
Mean encystment intensity (Gtrout) on YOY in Hiån, 2007 (larvae gill1, n = 20)
Mean encystment intensity (Gtrout) on older trout in Hiån, 2007 (larvae gill1, n = 13)
Microscope counts 0.0029 L + 0.58 (regression), 0.85, <0.05 26.8 L1.0 (Exp., base 10), 0.89, <0.05 0.78 e0.01L (Exp., base e), 0.89, <0.05 0.28 Ln(L) + 1.58 (natural log), 0.86, <0.05 0.01 W + 0.41 (regression), 0.82, <0.05 0.55 W0.34 (Exp, base 10), 0.88, <0.05 0.42 e0.037W (Exp, base e), 0.87, <0.05 0.095 Ln (W) + 0.49 (nat. log), 0.85, <0.05 Gmean photo/Gmean gill arch 1 (Mean prop of larvae on photo)
26.3 ± 5.8 24.4 ± 6.4 25.1 ± 6.7 24.8 ± 6.6 24.6 ± 6.5 24.3 ± 6.4 25.2 ± 6.8 24.6 ± 6.5 24.8 ± 6.6 29.7 ± 7.7
72.0 ± 27.1 77.5 ± 28.0 73.6 ± 26.3 74.0 ± 26.5 73.3 ± 26.3 73.4 ± 26.8 74.0 ± 26.4 75.1 ± 27.2 73.9 ± 26.5 73.9 ± 26.5
Older trout
r
p
r
p
– 0.95 0.94 0.94 0.94 0.95 0.94 0.95 0.94 0.95
– <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
– 0.98 0.98 0.98 0.98 0.99 0.98 0.99 0.98 0.97
– <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
of the spatula area, and C is the proportion of larvae per gill area for the gill region outside of the spatula area. This resulted in Q = 0.77 for YOY trout and Q = 0.76 for older trout from Hiån 2007. In 2008, Q = 0.87 for YOY trout, and Q = 0.89 for older trout. M is the estimate of the proportion of the spatula area on the photo in relation to the total area of the 1st gill arch for each trout. To calculate M, weight and length of the fish were related to the proportions of the numbers of gill filaments in the spatula area. Four equations with length and four with weight as the independent variables were tested based on linear regression, natural logarithm, exponential equation with base 10, and exponential equation with base e. This resulted in eight equations, which were all significant (M in the formula; Table 1). To obtain mean larval densities per gill, the number of larvae on the first gill arch also needed to be related to the mean number of larvae per gill arch. This was done by dividing Gmean all gill arches, which is the mean number of larvae on all gill arches, and Gmean gill arch 1, which is the mean number of larvae on the first gill arch. Lastly, a ninth equation used the proportion between the mean number of larvae at the spatula area on the photo, Gmean photo, and the mean number of larvae per gill, Gmean gill arch 1 instead of R/P. In this case, Gtrout, was calculated by the formula: Gtrout ¼ ðGphoto K Gmean photo =Gmean gill arch 1 Þ Gmean all gill arches =Gmean gill arch 1 :
The effect of subjecting the fish to the photograph method on trout survival and growth was tested in an aquaria experiment. Hatchery reared YOY trout from the Gullspång River were used in this experiment. Three different treatments, using 10 trout individuals per treatment, were compared. In the first treatment, the fish were tagged with PIT tags inserted into the peritoneal cavity, were measured for length and weight, and subjected to a simulated photo session by placing the spatula between the first and the second gill arch for one minute. This length of time was not exceeded in the field, and thus considered the maximum stress any individual fish experienced out of the water. The second treatment included PIT-tagging (Trovan microtransponder 101, 11.5 mm) and measures of length and weight. The third treatment was a control where only length and weight were measured. All trout were anesthetized with tricain methane-sulfonate (MS222) before the treatments were performed. The PIT-tag insertion treatment was tested as this is often used to mark trout in experiments, and was expected to increase the stress experienced the trout as compared to a treatment without PIT-tagging, as in the field investigation. The aquaria experiment was performed from 2008-11-10 to 2008-12-23 in 100 L aquaria with EHEIM 2217 pumps and standard filters. Temperature was measured every hour with a
HOBO Pendant Temperature/Light Data Logger. The mean water temperature was 11.2 ± 0.5 (SD) °C over the experimental period. Approximately half of the water in the aquaria was exchanged once a week. The trout were fed 4% of their body weight five times a week with defrosted chironomid larvae. Weight and length measures were performed every 2nd week on trout after anesthetizing with tricain methane-sulfonate (MS222). The relationship between the number of larvae in the spatula area on the photo and the actual number of larvae in the spatula area counted under microscope, and the relationship between the number of larvae per gill based on the photo-method and the numbers of larvae based on counting the larvae under microscope were analyzed using correlations. The relationship between the mean number of larvae per gill in each stream based on the photo-method and the numbers of larvae based on counting the larvae under microscope was also analyzed using correlations. The differences in mean larval encystment intensity in the streams based on the photo-method and mean larval encystment intensity based on counting the larvae under microscope were compared using paired t-tests. The differences in length, weight, length change and weight change in the aquaria experiment were analyzed using ANOVA:s. All measures of variation in the results section are given as ±1 SE. 3. Results There were significant correlations between the number of larvae in the spatula area counted on the preserved gills under the microscope and the number of larvae at the spatula area counted on the photograph on the trout from Hiån in October 2007 (Fig. 2a) and on every trout from all streams in June 2008 (Fig. 2b). 3.1. Encystment on individual brown trout in spring 2007 All correlations between encystment intensity based on the counted numbers of larvae under the microscope and encystment intensity estimated by using the photo-method for YOY and older in 2007 were significant (Table 1). Significant correlations with the highest r-values are shown for YOY and for older trout (Fig. 3). The mean encystment intensity was 26.3 ± 5.8 larvae gill1 and 72.0 ± 27.1 larvae gill1 on the YOY and older trout from Hiån in October 2007 respectively, based on the counted numbers of larvae under the microscope. These encystment rates did not differ significantly from the mean encystment of larvae based on the photo-method from Hiån in October 2007, which were between
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The mean difference and the absolute difference between encystment intensity based on the microscope counts and the photo-method in 2007 was 2.0 ± 2.3 larvae gill1 and 6.9 ± 1.7 larvae gill1 for YOY trout, and 1.4 ± 4.2 larvae gill1 and 10.4 ± 2.9 larvae gill1 for older trout. Seven of the 20 YOY trout individuals and four of the thirteen older trout individuals from 2007 lacked larvae on the spatula area, but were found to have low encystments intensities when counted under the microscope. The mean encystment intensity of these ‘‘zeros’’ was 1.5 ± 0.9 larvae gill1 on YOY trout and 9.6 ± 5.6 larvae gill1 and on older trout in 2007 when counted under the microscope. 3.2. Encystment on individual brown trout in spring 2008 Fig. 2a. Correlation between encystment intensity at the spatula area counted in microscope and encystment intensity at the spatula area counted on the photo on trout from Hiån autumn 2007 (r = 0.95, p < 0.0001, n = 25).
Fig. 2b. Correlation between encystment intensity at the spatula area counted in microscope and encystment intensity at the spatula area counted on the photo on trout from all streams spring 2008 (r = 0.99, p < 0.0001, n = 126).
There were significant correlations between encystment intensity based on microscope counts and the photo-method for both YOY and older trout in 2008 (Table 2). The correlation between the estimates of the encystment intensity based on the photomethod and the microscope counts had the highest r-values when using the estimation of P based on the weight and exponential equation for YOY trout, and P based on length and regression equation for older trout (Fig. 4). The mean encystment intensity on 0+ trout was 36.4 ± 6.1 larvae gill1 in 2008 based on microscope counts, and did not differ significantly from the photo-method, which was between 35.2 ± 5.9 and 39.0 ± 6.6 larvae gill1 (paired t-tests, p > 0.05). Gtrout based on the natural logarithm length equation (36.6 ± 6.2 larvae gill1) was closest to the mean encystment intensity based on microscope counts for YOY trout. The mean encystment intensity on older trout was 12.9 ± 4.2 larvae gill1 in 2008 based on microscope counts, and did not differ from the mean encystment intensity when based on the photo-method, which was between 12.2 ± 4.0 and 15.0 ± 4.9 larvae gill1 (paired t-tests, p > 0.05). Gtrout based on Gmean photo/Gmean gill arch 1 (12.2 ± 4.0 larvae gill1) was closest to the Gtrout based on microscope counts (Table 2). The mean difference and the absolute difference between encystment intensity based on microscope counts and the photomethod was 2.5 ± 1.4 larvae trout1 and 6.7 ± 1.1 larvae trout1 for YOY trout, and 0.72 ± 1.2 larvae trout1 and 4.4 ± 1.0 larvae trout1 for older trout in 2008 respectively. Six of the 63 YOY trout individuals and 12 of the 64 older trout individuals from 2008 that lacked larvae on the photo were found to have larvae when counted under the microscope. The mean encystment intensity of these ‘‘zeros’’ was 2.1 ± 1.3 larvae gill1 and 2.7 ± 1.0 on YOY and older trout respectively in 2008 when counted under the microscope. 3.3. Survival and growth of brown trout in aquaria experiment
Fig. 3. Correlations between the encystment intensity based on counting the larvae in microscope and based on the photo-method for YOY trout (squares; r = 0.95, p < 0.0001, n = 25) and for older trout (triangles; r = 0.99, p < 0.0001, n = 13) in 2007. The estimation of P for YOY and older trout is based on the proportion of filaments on the spatula area at the photo, and the exponential equation with the base e and weight, respectively.
24.3 ± 6.4 larvae gill1 and 29.7 ± 7.7 larvae gill1 on YOY trout and between 73.3 ± 26.3 larvae gill1 and 77.5 ± 28.0 larvae gill1 on older trout (paired t-tests, p > 0.05). The mean encystment intensity of larvae based on the formula using the estimation of P based on length and exponential equation with the base 10 (25.1 ± 6.7 larvae gill1) and based on length and natural logarithm equation (73.3 ± 26.3 larvae gill1) were closest to mean encystment of larvae for YOY trout and for older trout respectively based on counted number of larvae under the microscope (Table 1).
Twenty-five out of 30 trout individuals survived the 30 days aquarium experiment. Eight trout individuals survived the treatment with a PIT-tag, length and weight measure, and the simulated photo-method. Nine trout individuals survived the treatment with a PIT-tag and length and weight measure, while eight trout individuals survived the control treatment with only length and weight measure. There was no difference in weight or length of the trout between the three treatments at any of the four occasions when the trout was measured (ANOVAs, p > 0.05). Likewise, there was no difference in weight or length change between the three treatments after 10, 20 and 30 days (ANOVAs, p > 0.05, n = 25) (Fig. 5). 3.4. Mean encystment intensity in the streams The mean encystment intensity varied between 0 and 119 ± 41 larvae gill1 and 0 and 105 ± 38–122 ± 46 larvae gill1 on YOY trout (n = 1–18) and between 0 and 132 (one ind.) larvae gill1 and 0 and
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Table 2 Methods used in the estimation of the proportion of the number of larvae at the spatula area on the 1st gill arch (M). Mean larval encystment of YOY in all streams 2008. Correlation between encystment intensity based on microscope counts and the photo-method in Hiån in October 2007. Mean larval encystment of older trout in all streams 2008. Method used in the estimation of the proportion of numbers of larvae at the spatula area on the 1st gill arch (M), r2, p
Microscope counts 0.0029 L + 0.58 (regression), 0.85, <0.05 26.8 L1.0 (Exp., base 10), 0.89, <0.05 0.78 e0.01L (Exp., base e), 0.89, <0.05 0.28 Ln(L) + 1.58 (nat. log), 0.86, <0.05 0.01 W + 0.41 (regression), 0.82, <0.05 0.55 W0.34 (Exp, base 10), 0.88, <0.05 0.42 e0.037W (Exp, base e), 0.87, <0.05 0.095 Ln (W) + 0.49 (nat. log), 0.85, <0.05 Gmean photo/Gmean gill arch 1 (Mean prop of larvae on photo)
Mean encystment intensity of YOY in all streams 2008, (larvae gill1, n = 62)
36.4 ± 6.1 35.2 ± 5.9 37.5 ± 6.4 36.9 ± 6.2 36.6 ± 6.2 35.9 ± 5.9 39.0 ± 6.6 36.6 ± 6.1 37.8 ± 6.4 36.9 ± 6.1
Fig. 4. Correlations between encystment intensity based on counting the number of larvae under the microscope and based on the photo-method for YOY trout (squares; r = 0.98, p < 0.0001, n = 62) and for older trout (triangles; r = 0.97, p < 0.0001, n = 64) in 2008. The estimation of P for YOY and older trout is based on the exponential weight equation with base 10, and length and regression, respectively.
Correlations of the number of larvae per gill on YOY trout based on microscope count and the photo-method (Gtrout) in all streams in May 2008 (n = 62) r
p
– 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.97
– <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Mean encystment intensity of older trout in all streams 2008, (larvae gill1, n = 64)
12.9 ± 4.2 13.7 ± 4.5 14.6 ± 4.8 14.5 ± 4.8 14.4 ± 4.7 14.7 ± 4.9 15.0 ± 4.9 14.6 ± 4.8 14.9 ± 4.8 12.2 ± 4.0
Correlations between encystment intensity on older trout based on microscope counts and the photo-method (Gtrout) in all streams in May 2008 (n = 64) r
p
– 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.96
– <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Fig. 5. Weight of brown trout at the beginning of the aquaria experiment (0 days), after 10, 20 and 30 days in the treatments (1) control (triangles), (2) pit-tag marked fish (squares), (3) pit-tag marked fish and photo-method (diamonds).
4. Discussion 136–160 larvae gill1 on older trout (n = 1–10) in the 16 streams in 2008 when based on the microscope counts and the photo-method, respectively. The mean encystment intensity in the streams were estimated to be zero based on the photo-method when there in fact were mean encystments of 0.9 ± 0 (one individual), 1.0 ± 0 (one individual) and 2.7 ± 3.3 (one of three individuals infected) larvae gill1 on YOY trout, and 0.1 ± 0.07 (one of four individuals infected) and 0.5 ± 0.3 (two of three individuals infected) larvae gill1 on older trout. In the other streams, there were no differences in the mean encystment per gill when based on microscope counts versus the calculations based on the photo-method (paired t-tests, p > 0.05). There were significant correlations between the mean encystment intensity in each stream based on microscope counts and the photo-method for YOY and older brown trout in June 2008. Encystment intensity based on the photo-method had the highest r-values when P was based on the exponential length equation with base 10 for YOY trout, and on Gmean photo/Gmean gill arch 1 for older trout, respectively (Fig. 6).
This investigation showed that a non-destructive method could be used instead of a destructive method when investigating the parasitic stage of a unionid mussel species on its host fish. The photo-method used here measured the encystment of M. margaritifera glochidia larvae on S. trutta gills without injuring the fish, or causing reduced growth of the fish. Therefore, it may be possible to investigate every life-stage of the highly threatened unionoid mussels such as M. margaritifera using non-destructive methods (Hastie and Boon, 2001; Hastie and Young, 2003; Österling et al., 2008, 2010). All equations relating the number of larval encystments at the spatula area to the number of larval encystments at the complete area of the 1st gill arch could be used for the estimation of the numbers of larvae on each trout individual. This resulted in significant correlations between the estimation of encystment intensity based on the photo-method and the microscope counts. Consequently, each one of the equations relating the number of larval encystments at the spatula area to the number of larval encystments at the complete area of the 1st gill arch could be used when
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Fig. 6. Correlation between mean encystment intensity per stream based on counting the number of larvae under the microscope and the mean encystment intensity per stream based on the photo-method on YOY trout (squares; r = 0.99, p < 0.0001, n = 13) and for older trout (triangles; r = 0.99, p < 0.0001, n = 16) in 2008. The estimation of P for YOY and older trout is based on the exponential equation with the base 10 and length, and proportion of filaments on the spatula area at the photo, respectively.
estimating the number of larvae on M. margaritifera using the photo-method. However, the usefulness of these equations may differ between M. margaritifera distribution areas. Therefore, the equations could be tested in M. margaritifera populations in other parts of its distribution, and on other unionid mussel species using salmonids as hosts, as the morphology of their gills resemble brown trout gills. On other mussel species, using other fish species as hosts, the likelihood of host-species-specific adjustments of the photo-method probably increase. The estimation of the encystment intensity on individual fish using the photo-method was significantly related to the encystment intensity on individual fish based on microscope counts. In fact, the correlations explained ninety-four percent or more of the variation in the analyses, and the slopes of the correlations were close to one. Only occasionally, the encystment intensity differed as much as about two times when comparing the photomethod with the method based on counting the larvae under the microscope. However, the estimates of the two methods were always in the same order of magnitude. The photo-method showed a high precision when estimating the mean encystment intensity per stream on both YOY and older trout, which is often needed when investigating population regulation and host age-dependent sensitivity of this life-stage (Young and Williams, 1983, 1984; Bauer, 1987b, 1987c; Österling et al., 2008). Therefore, the photo-method may be applied when estimating mean encystment intensity in different mussel – brown trout streams. In fact, the photo-method seemed to be reliable even when there were small sample sizes (i.e. low brown trout density), which was the case for several of the individual streams. However, the estimation of the mean encystment intensity based on the photo-method was zero in a few occasions when there in fact were a few encystments based on counting the larvae under the microscope. This was mainly a result of few encystments on few trout individuals in streams with low mussel density which was missed on the relatively small area covered by the photographs. The mean encystment intensity in these measurements was only between 0.1 and 2.7 larvae gill1, which is a very low contribution to the population (Österling et al., 2008). However, the estimated encystment intensity based on the photo-method was never zero when there were at least five trout individuals. Often there is a possibility to catch five or more trout individuals when electro-fishing in M. margaritifera streams (Hastie and Boon, 2001; Hastie and Young, 2001; Geist et al., 2006; Österling et al., 2008). Therefore, it should be possible to capture enough trout individuals to make appropriate measurements of mean encystment intensity.
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The estimation of encystment intensity was slightly better in spring than in the autumn, probably because the larger larvae in the spring (Hastie and Young, 2001, 2003) were more easily recognized on the photo compared to the smaller larvae in the autumn. However, the strength of these estimations was generally high both in the autumn and in spring. Thus, it may also be possible to estimate changes in the encystment intensity between mussel and trout populations, and differences between different year classes of brown trout without harming the fish. In fact, it may even be possible to measure changes in encystment intensity on the individual fish, as the survival rates were high when marked with PIT-tags. Lastly, it would also be possible to establish relationships between the numbers of encysted larvae present in relation to the density of the mussel population within the stream. There are a number of applications in which this photo-method may be used when managing threatened unionoid mussel populations (Geist, 2010), apart for measuring mean encystment intensity. First, the photo-method may be used to select fish with high numbers of infested larvae. This may be needed at streams where cultivation measures are performed, as fish density may be a limiting factor both in streams and at breeding facilities. Fish with high larval load may therefore be selected to raise the numbers of juvenile mussels (Österling et al., 2008). Secondly, the photo-method combined with PIT-tagging did not seem to have any negative effects on growth or survival of the trout. Therefore, combining the photo-method with PIT-tagging may allow studies of changes in encystment intensity on individual fish. It would also be possible to study colonization distances of the mussel, if trout are marked and photographed at both capture and recapture events. Lastly, the photo-method could be tested for other mussel species, as the larvae of M. margaritifera are among the smallest of all unionid mussels and therefore less easy to detect compared to mussel species with larger glochidia larvae (Bauer, 1987a, 1998). If the photo-method is applicable to other unionid mussel species, it should be possible to investigate where in the life-cycle several unionid species are threatened without using harmful methods at all, as the juvenile and adult stages can already be investigated using non-harmful methods (Hastie and Boon, 2001; Young et al., 2001; Hastie and Young, 2003; Österling et al., 2008, 2010). Acknowledgments I thank Fortums Nordiska Miljöfond for financing this study, H. Söderberg for inspiration, H. Karlsson for field assistance, and B. Arvidsson, L. Greenberg and four anonymous referees for commenting earlier versions of the manuscript. The electro-fishing and all other fish handling was performed under licenses from the Country Administration in Västernorrland and from the Swedish Animal Welfare Agency CFN, Gothenburg (Dnr: A81-07). References Awruch, C.A., Frusher, S.D., Pankhurst, N.W., Stevens, J.D., 2008. Non-lethal assessment of reproductive characteristics for management and conservation of sharks. Marine Ecology – Progress Series 355, 277–285. Bauer, G., 1987a. Reproductive strategy of the freshwater pearl mussel Margaritifera margaritifera. Journal of Animal Ecology 56, 691–704. Bauer, G., 1987b. The parasitic stage of the freshwater pearl mussel (Margaritifera margaritifera L.) 2. Susceptibility of brown trout. Archiv Fur Hydrobiologie 76, 403–412. Bauer, G., 1987c. The parasitic stage of the freshwater pearl mussel (Margaritifera margaritifera L.) 3. Host relationships. Archiv Fur Hydrobiologie 76, 413–423. Bauer, G., 1998. Allocation policy of female freshwater pearl mussels. Oecologia 117, 90–94. Bauer, G., Vogel, C., 1987. The parasitic stage of the freshwater pearl mussel (Margaritifera margaritifera L.) 1. Host response to glochidiosis. Archiv Fur Hydrobiologie 76, 393–402. Bogan, A.E., 1993. Fresh-water bivalve extinctions (Mollusca, Unionoida) – a search for causes. American Zoologist 33, 599–609.
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