Project Title: Delta Smelt Acoustic Tag Development introduction



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Project Title: Delta Smelt Acoustic Tag Development
INTRODUCTION

Delta smelt, previously one of the most common resident pelagic fish in the San Francisco Bay-Delta Estuary, have dramatically declined in abundance since the 1980s and were listed as threatened under the Federal and California Endangered Species Acts (ESA) in 1993. Many, if not all ecological questions surrounding the decline, protection, and recovery of delta smelt require knowledge of life history strategies, migration and spawning behavior, habitat usage, and exposure to key environmental stressors. To date, little is known about the spatial and temporal distribution of delta smelt in the San Francisco Bay-Delta Estuary. This research project aims to assess the feasibility of tagging adult delta smelt to advance the long-term goal of establishing a Bay-Delta system-wide smelt acoustic telemetry system.

As outlined under the ‘Scope of Work’ section in the “Development of an Acoustic Transmitter Suitable For Use In Delta Smelt” Cooperative Agreement between Bureau of Reclamation and University of California, Davis, three major tasks are to be performed throughout the Delta Smelt Acoustic Tag Development study. Key objectives for this project are to 1) determine the body cavity size and shape of adult delta smelt; 2) determine the size and weight constraints of acoustic transmitters for successful implantation in delta smelt; and 3) prepare a written report detailing the methods used and the principal findings and give a presentation of the findings at the Interagency Ecological Program (IEP) annual workshop. This interim progress report is intended to inform the sponsor about advancements to-date and scheduled activities for the remainder of the project period.
ACTIVITIES AND ACCOMPLISHMENTS TO-DATE

Adult delta smelt body cavity size and shape

To gain information needed to establish transmitter size constraints, the size and shape of the peritoneal cavity, into which acoustic tags are usually inserted, was investigated. For this task, 20 two-year-old delta smelt were transported from the Fish Conservation and Culture Lab (FCCL) to a laboratory in the Department of Civil and Environmental Engineering at UC Davis in February 2011. Single fish were removed from the carboy used for transportation, euthanized with an overdose of the anesthetic MS222, and their weight and length were measured (Table 1).



Table 1. Averages (AVE) and standard deviations (StDev) of length (cm) and weight (g) measurements of twenty two-year-old delta smelt, and of measurements from casts extracted from the peritoneal cavity. Cast weight (g), volume (ml), length (cm), maximum height and width (cm), and distance of area of maximum height and width from anterior end of cast are displayed. Fish were grouped by gender.





Length (cm)


Weight (g)


Cast (g)


Cast (ml)

Cast length (cm)

Cast max. height (cm)

Cast max. width (cm)

Distance from anterior

Male delta smelt (AVE)

8.89

6.45

0.29

0.29

3.82

0.89

0.64

1.41

StDev

0.38

0.77

0.10

0.09

1.02

0.07

0.05

0.42

Female delta smelt (AVE)

9.51

8.88

0.32

0.33

3.13

1.11

0.78

1.53

StDev

0.61

1.84

0.05

0.05

0.45

0.06

0.10

0.42

Subsequently, fish were injected with a polyurethane resin (EasyFlo 60 Liquid Plastic) into the peritoneal cavity. A low viscosity resin was chosen to obtain a bubble-free, detailed cast of the peritoneal cavity. After approximately 15 minutes, the solidified plastic was recovered by making an incision along the peritoneal cavity of the fish and extracting the cast (Figure 1).


Figure 1. Adult delta smelt with extracted cast.

060.jpg

Various cast measurements important for transmitter design were taken of all 20 casts (Figure 2):



  1. Cast weight (g) and volume (ml), determined by water displacement.

  2. Cast length (cm); and

  3. Maximum height and width of cast, and distance of point of maximum height and width from anterior end of cast.

Furthermore, three-dimensional images of all casts were taken with a CT scanner at a resolution of 50 micron voxel. Image analyses have not been completed to-date.
Figure 2. Measurements taken on casts from the peritoneal cavity of adult delta smelt: cast length (A), maximum height (B), and distance of point of maximum height from anterior end of cast (C). Not shown is the measurement of maximum width of the cast. The widest part of the cast was typically found where maximum cast height occurred.

cast measurements.jpg
The same procedures were applied to 20 one-year-old delta smelt, and results are presented in Table 2. The average weight and length of two-year-old delta smelt was greater than that of one-year-old fish. Furthermore, the average weight and length of females from both age-groups was greater than that of the respective male fish. In accordance with these findings, cast measurements indicated that two-year-old delta smelt have larger peritoneal cavities in comparison to one-year-old delta smelt, and that females have larger peritoneal cavities than male fish. Overall, peritoneal cavity dimensions of both delta smelt age groups permitted the use of the smallest currently available acoustic transmitter, the JSATS (Juvenile Salmon Acoustic Telemetry System) tag, as a prototype for production of dummy tags along a gradient of sizes and weights. The JSATS-tag represented the upper size limit for dummy-tags to be tested in upcoming tag effect studies, while a medium and small sized tag had to be designed as part of this study.
Table 2. Averages (AVE) and standard deviations (StDev) of length (cm) and weight (g) measurements of twenty one-year-old delta smelt, and of measurements from casts extracted from the peritoneal cavity. Cast weight (g), volume (ml), length (cm), maximum height and width (cm), and distance of area of maximum height and width from anterior end of cast are displayed. Fish were grouped by gender.





Length (cm)


Weight (g)


Cast (g)


Cast (ml)

Cast length (cm)

Cast max. height (cm)

Cast max. width (cm)

Distance from anterior

Male delta smelt (AVE)

7.02

2.80

0.21

0.20

3.13

0.70

0.52

1.07

StDev

0.42

0.58

0.08

0.08

0.61

0.11

0.08

0.29

Female delta smelt (AVE)

7.13

3.29

0.23

0.23

3.50

0.83

0.65

1.32

StDev

0.34

0.44

0.09

0.10

0.17

0.05

0.05

0.18


Dummy-tag design

Dummy tags designed and tested during this study are shown in Figure 3. The design of dummy tags along a gradient of sizes was based on a) the average size of the peritoneal cavity of delta smelt determined by cast measurements, b) a literature review of acceptable tag burden (i.e., the weight of a transmitter relative to the weight of the fish), and c) the smallest acoustic transmitter currently available commercially. Given the peritoneal cavity size and shape of both two-year-old and one-year-old delta smelt, we decided to use the JSATS microtransmitter as the large sized dummy tag for testing in delta smelt. However, due to the high average tag burden of approximately 14% for one-year-old delta smelt inserted with the JSATS-tag, we removed one of the two batteries to lower the weight of the tag from 0.433g to 0.308g, which lowered the tag burden to about 10%.


Figure 3. Commercially available tags (two tags on the left) and newly designed dummy-tags (two tags on the right) inserted into one-year-old delta smelt during a 28-day survival study. From left to right: PIT (Passive Integrated Transponder) tag (0.100 g), JSATS (Juvenile Salmon Acoustic Telemetry System) tag (0.308 g), medium sized dummy tag (0.121 g), and small sized dummy tag (0.094 g). One of the two batteries was removed from the JSATS-tag to decrease the weight of the microtransmitter.
all tags.jpg

Next, we created a medium sized tag prototype approximately 25% smaller by volume than the JSATS microtransmitter. The medium sized dummy-tag was produced by initially carving out a cavity in modeling clay with the desired dimension of the dummy-tag, and filling the cavity with polyurethane. Once the polyurethane hardened, it was removed from the modeling clay and was subsequently used as the dummy-tag. The same procedure was used to fabricate a small sized tag prototype with approximately 25% less volume than the medium sized tag. Approximately 60 tags of each sized dummy-tag were produced. To increase the weight of the tags, short pieces of steel wire were incorporated into the polyurethane resin. Each tag received an individual code using a solvent resistant marker (Figure 3). The average weights of the medium and small tags were 121.3 mg and 94.5 mg, respectively, representing a tag burden for average one-year-old delta smelt of approximately 3 g of 4.0% and 3.1%, respectively.


Design of a recirculating aquaculture system for delta smelt tagging experiments

To determine effects of the three differently sized tags and the implantation procedure for delta smelt, a recirculating aquaculture system with a total volume of about 10,000 liters of water was built (Figure 4). Our aim was to replicate the recirculating systems used for delta smelt cultures at the FCCL. The basic components of the recirculating system built at the Center for Aquatic Biology and Aquaculture (CABA) at UC Davis include eight fish tanks, a heat pump for water temperature regulation, a floating beat filter to trap solid waste and remove unwanted and toxic nitrogenous waste, a system reservoir, pumps, piping, fittings and other structural elements.


Figure 4. Three dimensional schematic diagram of the reciruclating aquaculture system.


By mid March, after completion of the recirculating aquaculture system, fish were introduced to test the system and to activate the biological filters. Approximately 600 one-year-old delta smelt were distributed equally between seven circular tanks (diameter 2 m; depth 0.76 m; inflow ~8 l/min, water velocity ~8 cm/s) supplied with non-chlorinated, air-equilibrated, temperature-controlled well water. One tank was left empty for specific purposes such as quarantine. Tanks were covered with fine screen mesh to minimize illumination and disturbance. Fish care followed the standard operating procedures established at FCCL for feeding, tank maintenance, water quality monitoring and target water quality parameter values, and stocking densities. Critical water quality parameters such as temperature, dissolved oxygen, pH, total ammonia nitrogen, and electrical conductivity were monitored using probes or chemical analyses.

Water temperature in all tanks remained consistently at the targeted 13˚C (±0.5) over the entire testing phase for the aquaculture system between March 17 and May 16. Dissolved oxygen, pH, and electrical conductivity remained within the predetermined ranges for each parameter. Concentrations of ammonia, a natural byproduct of fish metabolism, initially increased during the first two to three weeks but never reached levels considered toxic to fish. Due to the low density of fish in our system, the establishment of bacteria in the biological filters to convert ammonia into harmless end products took approximately 40 days. During the first 30 days of the two-month testing phase, mortality was approximately 9%, higher than the expected 5% (personal communication, FCCL), but dropped to anticipated levels of 5% in the second month. The elevated mortality of 9% was attributed to the initially increased levels of nitrogen. After the biological filtration system equilibrated, tag and procedure effects on delta smelt were evaluated.


Biological effects of tag implantation in one-year-old delta smelt

To assess possible effects of the surgical procedure and/or the tag itself, a 28-day experiment was designed to evaluate the survival, growth, tag retention, and wound healing of one-year-old delta smelt. On May 17, after successfully holding adult delta smelt for two months, a 28-day experiment was initiated with four treatment and three control groups with 60 fish per treatment and control group (420 fish total).

All groups, with the exception of one control group (Control; not handled or tagged) were anesthetized and body mass and fork length were measured to determine growth during the experiment. For treatment groups receiving the JSATS-tag, medium sized dummy-tag, or small sized dummy-tag, a small incision (~5mm) was made close to the linea alba, and the tag was inserted into the coelomic cavity. Incisions were closed with an absorbable monofilament. The PIT-tag treatment group received an even smaller incision (~3mm) for tag implantation, and the opening was left untreated. To control for handling and anesthesia effects, one control group was anesthetized and only measured (MS222 Control), while a third control group was anesthetized, measured, and received an incision/suture but no tag to determine effects of the tagging procedure itself (Suture Control).

Cumulative mortality of fish from all treatment and control groups over the 28-day study period is displayed in Figure 5 and discussed below. Growth, tag retention, and wound healing data is currently being analyzed. All three control groups experienced similar numbers of mortalities over the duration of the study period. Mortality of all fish implanted with tags was significantly higher than that of the control groups, and survival was a function of tag size/weight. In the untreated control group, 46 delta smelt (77%) survived, while 52 (87%) and 37 (62%) fish survived from the MS222 and Suture Control groups, respectively. Among the treatments, 34 (57%) delta smelt survived in the PIT-tag group, 22 (37%) in the small sized dummy-tag group, 11 (18%) in the medium sized dummy-tag group, and 4 (7%) in the JSATS-tag group.


Figure 5. Percent cumulative mortality of one-year-old delta smelt tagged with large, medium, or small tags, Passive Integrated Transponder (PIT)-tags, and untagged control groups over 28 days. The large tag consisted of a JSATS tag with one battery removed, while the medium and small tags were made out of polyurethane and steel wire. Controls consisted of fish not handled or tagged (Control), handled fish (anesthetized and measured; MS222), and handled fish with incision and wound closure but without insertion of tag (Suture Control). All tagged treatment groups and the Suture Control group received incisions, which were closed with an absorbable monofilament, except for the PIT-tag group where incisions were left untreated.
all treatments 28 days.wmf

Statistical comparison of the Control and MS222 groups showed no significant difference in cumulative mortality over 28-days, indicating that handling and anesthesia did not affect the survival of delta smelt during the study period. Mortality of fish with surgery but no tag (Suture Control) did not differ significantly from that of the Control group for most of the study period (days 1 through 24), suggesting that the surgical procedure did not contribute significantly to mortality in fish receiving a tag. Results from the tagged treatment groups indicate that a) overall cumulative mortality increases with tag size/weight, b) highest mortality rates occur in the first week and then level off between days 8 and 12, and c) time spent for suturing the wound does not increase mortality significantly in comparison to fish with untreated incisions.

We believe that various factors potentially compromising survival in this first experiment can be either greatly reduced or eliminated in subsequent experiments performed as part of this study. First, overall cumulative mortality of all groups after 28 days was higher than anticipated. This was due to a second increase in mortality approximately 18 to 20 days after the study was initiated, and it affected all treatment and control groups similarly (Figure 5). Mortality during the first few days after surgery was expected due to adverse effects on fish from handling, wound healing processes, and possible bacterial infection. However, similar studies with other fish species have shown that incisions are mostly closed before or by day 14, and little change in incision openness occurs beyond this point. Closure of the incision decreases the risk of wound infection considerably, and thus mortality related to the surgery itself declines. In accordance with these findings, mortality leveled off between days 8 and 12 and survival of the Large, Medium, and Small tag treatment groups was roughly 30%, 50%, and 75%, respectively, after two weeks. Investigation into possible causes for the second increase in mortality revealed that diurnal water temperature variation, the daily difference between the high and low water temperature in holding tanks, increased considerably starting approximately 20 days after the experiment was initiated, coinciding with mortality rate increases of all treatment and control groups. The diurnal water temperature variation increase was due to the seasonal increase of air temperature. Water temperature differences of up to 3°C were recorded (Figure 6), which likely lead to temperature stress which has dramatic effects on the metabolism of fish, including their immune system. To avoid temperature stress in the future, the remaining tagging studies will be conducted between November and January, when air temperatures will be much closer to the required water temperature and heat pumps will be able to keep diurnal temperature variation below 1°C.
Figure 6. Diurnal water temperature variation during the 28-day survival study of tagged and control groups of one-year-old delta smelt. Water temperature was measured every 10 minutes with a temperature data logger emerged in a biofilter. Shown are daily variation in water temperature between the highs and lows of a 24-hour period, typically occuring during late afternoons and early mornings, respectively.

diurnal temperature variation.wmf

Second, building, testing, and priming the recirculating system delayed the start of the first tagging experiment, and the start of the study in May coincided with the peak of the delta smelt spawning season. A high percentage of delta smelt females used in the tagging experiment were gravid, while most male gonads were full of sperm. Gonads are located in the peritoneal cavity, thus decreasing space available for a tag considerably. In addition, it is likely that gravid fish react differently and are more vulnerable to negative effects of the tagging procedure and the tag itself. To avoid tagging fish in a reproductive stage, the remaining tagging studies are planned for late fall and early winter when delta smelt are in pre-spawn condition, allowing the evaluation of tag and tagging effects for non-gravid fish.

Third, it came to our attention during the two month holding phase and during the tagging experiment, that higher mortality rates typically followed bead filter backwashing events. Trapped particulates from fish food and feces collect in the bead filter over time, and beads stick together by the waste material. Filter backwashing removes the waste and frees the beads. The filter in our system was flushed roughly every three weeks, and it was observed that filter backwashing was followed by: (a) elevated levels of ammonia during the next two to three days and (b) increased mortality. For example, the bead filter was flushed on June 1 (day 16) during the tag effect study, and mortality started increasing between days 17 and 20 for all groups. In our future tagging experiments with one-year-old and two-year-old delta smelt in the fall and winter 2011/12, we will flush bead filters daily to prevent accumulation of particulates in the filter to a point where flushing frees enough waste to increase ammonia concentrations to harmful levels.
Remaining activities

1. Survival studies with non-gravid two-year-old delta smelt and non-gravid one-year-old delta smelt during fall and winter 2011-2012.



2. Presentation of our findings at the next IEP annual workshop. Poster presentation of results from this study at the ‘State of the San Francisco Estuary 10th Biennial Conference’ in Oakland, CA. An abstract was submitted July 20, 2011.

3. Final report.

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