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North American Journal of Aquaculture

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Culture of Tubifex tubifex: Effect of Feed Type, Ration,

Temperature, and Density on Juvenile Recruitment,
Production, and Adult Survival
a a a
Randall W. Oplinger , Matt Bartley & Eric J. Wagner
a
Utah Division of Wildlife Resources , Fisheries Experiment Station, 1465 West 200 North,
Logan, Utah, 84321, USA
Published online: 02 Feb 2011.

To cite this article: Randall W. Oplinger , Matt Bartley & Eric J. Wagner (2011) Culture of Tubifex tubifex: Effect of Feed
Type, Ration, Temperature, and Density on Juvenile Recruitment, Production, and Adult Survival, North American Journal of
Aquaculture, 73:1, 68-75

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 North American Journal of Aquaculture 73:68–75, 2011


C American Fisheries Society 2011

ISSN: 1522-2055 print / 1548-8454 online

DOI: 10.1080/15222055.2010.549028

ARTICLE

Culture of Tubifex tubifex: Effect of Feed Type, Ration,

Temperature, and Density on Juvenile Recruitment,
Production, and Adult Survival

Randall W. Oplinger,* Matt Bartley, and Eric J. Wagner

Utah Division of Wildlife Resources, Fisheries Experiment Station, 1465 West 200 North, Logan,
Utah 84321, USA
Downloaded by [Mississippi State University Libraries] at 19:43 15 October 2013

Abstract
The oligochaete worm Tubifex tubifex is widely cultured as a fish food. Stocking of certain T. tubifex strains has
been shown to help prevent the occurrence or reduce the severity of whirling disease caused by Myxobolus cerebralis.
Optimal culture conditions (e.g., temperature, density, and feed type) are not known for T. tubifex. We conducted four
experiments to improve our knowledge of culture methods for T. tubifex. First, we evaluated the survival, growth,
and recruitment of T. tubifex that were fed one of three different diets. We found that the performance of worms fed
cow manure was poor and that growth and recruitment were best when they were fed either a commercial fish-flake
food (Tetramin) or a commercial sinking fish feed containing spirulina Spirulina spp. In the second experiment, we
evaluated the performance of worms fed Tetramin at rations of 0, 2.5, 5.0, and 10.0% of body mass/d and found the
greatest growth and recruitment at the 5.0% and 10.0% rations. In the third experiment, we found high growth and
survival among T. tubifex at temperatures between 12◦ C and 27◦ C; however, recruitment decreased at temperatures
above 21◦ C. In the final experiment comparing seven initial adult stocking densities (2,675–267,451 individuals/m2),
juvenile recruitment and the net increase in biomass were found to be highest at the lowest initial density of 2,675
adults/m2. Recruitment decreased significantly at densities above 6,686 adults/m2.

The oligochaete worm Tubifex tubifex is widely distributed
throughout the world (Kathman and Brinkhurst 1998; Zendt
and Bergersen 2000), tolerating a wide variety of environmen-
tal conditions. Its absence, however, can be indicative of water
pollution (Brinkhurst and Gelder 1991). Algae, decaying or-
ganic material, and microbes constitute major components of
their diet (Brinkhurst and Gelder 1991). Due to its tolerance
of a variety of environmental conditions and the high densities
that it can attain (Bonacina et al. 1989a, 1989b; Steinbach El-
well et al. 2006), T. tubifex is widely cultured and marketed as a
food for aquarium fish. Unfortunately, very little is known about
the optimal culture conditions (e.g., temperature, density, feed
type, etc.) for T. tubifex. Previous studies have raised them on
feed consisting of wheat bran, mustard oil cake, cow manure,
lettuce, and spirulina (blue-green algae in the genus Spirulina)
(Ahamed and Mollah 1992; Bonacina et al. 1989a). The opti-
mal ration, temperature, and density for T. tubifex culture has
not been documented.
Stocking of certain T. tubifex strains may help prevent
the occurrence or reduce the severity of whirling disease, a
salmonid disease caused by the myxosporean Myxobolus cere-
bralis (Beauchamp et al. 2006). Tubifex tubifex plays a critical
role in the transmission of this disease, which has caused signif-
icant declines in some western North American populations of
rainbow trout Oncorhynchus mykiss (Markiw and Wolf 1983;
Nehring and Walker 1996; Vincent 1996). When an infected fish
dies, it releases myxospores into the wild. These myxospores are
ingested by T. tubifex and attach to their gut epithelium. After
further development, T. tubifex sheds triactinomyxons (TAMs)
into the water, and these TAMs attach to and eventually infect

*Corresponding author: randyoplinger@utah.gov

Received June 16, 2010; accepted September 4, 2010
Published online February 2, 2011

68
 CULTURE OF Tubifex tubifex
a fish host (MacConnell and Vincent 2002). Within T. tubifex,
there are multiple strains or lineages. Some of these lineages
(I and III) are susceptible to the parasite and produce TAMs,
whereas lineages V and VI deactivate M. cerebralis myxospores
and do not produce TAMs (Beauchamp et al. 2002; DuBey
et al. 2005). Laboratory research indicates that the stocking of
T. tubifex strains resistant to M. cerebralis could be used to re-
duce the incidence of whirling disease or even eradicate it from
the wild. The basic theory is that in an infected waterway that
harbors a population of a susceptible strain of T. tubifex, lineage
I or III, can be augmented or replaced by resistant strain, lineage
V or VI (Steinbach Elwell et al. 2006).
The success of a resistant-strain stocking program hinges on
the ability to successfully culture large quantities of T. tubifex.
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Many authors report success at rearing T. tubifex in a laboratory
(e.g., Beauchamp et al. 2006; Rasmussen et al. 2008; Kerans
et al. 2005), but these studies have simply maintained stocks
and have not determined the conditions necessary to maximize
growth and reproduction. The mass culture of T. tubifex, how-
ever, will require optimization of rearing conditions. The ob-
jective of our study was to improve our understanding of the
environmental conditions that optimize the culture of T. tubifex.
We outline the results of four studies that evaluated the optimal
feed type, ration, temperature, and density for rearing T. tubifex.
The results from our studies should aid in the development of
resistant-strain stocking programs and could help increase the
efficiency of production for the aquarium trade.
METHODS
The T. tubifex used in our experiments were provided by
the Colorado Division of Wildlife and were collected by hand
from two sources: Mount Massive Wilderness, Colorado (100%
lineage III), and White River, Colorado (2% lineage I, 14%
lineage III, and 84% lineage VI). Lineage determinations were
performed by Pisces Molecular (Boulder, Colorado) using poly-
merase chain reaction (PCR) assays (Beauchamp et al. 2002;
Nehring 2008). The worms were shipped to the Utah Division
of Wildlife Resources Fisheries Experiment Station in Logan
where they were stored for 6–12 months prior to experiment
initiation. During this time, they were stored at 14◦ C on a 12 h
light : 12 h dark photoperiod in a plastic tub filled with water
and sediment. They were fed ad libitum with a feed mix de-
veloped by Barry Nehring of the Colorado Division of Wildlife
(15.7% Algamac 2000, 32.8% Tetra Colorfin, and 51.4% de-
hydrated spirulina disks). The PCR assay used to determine
T. tubifex strains analyzed samples consisting of multiple indi-
viduals from which the percentage of DNA attributed to each
lineage was determined. Since we could not identify the lineage
of individual worms, it was not possible to determine whether
the growth and production of individuals was dominated by a
single strain. The original goal of this experiment was to com-
pare the effect of feed type, ration, temperature, and rearing
density between individuals from lineages III and VI. Because
69
of these analytical limitations and the fact our cultures were not
pure, we decided to use T. tubifex from the Mt. Massive source
for half of our replicates, and in other half of the replicates we
used individuals from the White River source. We did not mix
individuals from the two sources.
For each of the four experiments, 400-mL plastic tri-point
beakers containing 300 mL of well water (pH = 7.2, hardness =
250 mg/L, total alkalinity = 190 mg/L) and 25 g of dried mud
collected from a local lake (25–106 µm in diameter, autoclaved)
were used to hold the worms. Fresh autoclaved sediment was
used at the start of each experiment. Aeration was provided
by an air stone connected to an aquarium air pump. Beakers
were covered with disposable, plastic weighing boats. A 1-cm-
diameter hole was cut into the middle of each weighing boat to
accommodate the air stones.
At the conclusion of each experiment, the contents of each
replicate beaker were sieved through a 180-µm mesh sieve.
Separate counts were made for juvenile and adult T. tubifex. The
two size-classes were easily distinguished because adults were
significantly larger than the juveniles. In situations where a large
number of T. tubifex were harvested, the sample was subdivided,
and half of the sample was randomly selected and counted. The
combined wet mass of juveniles and adults was measured to the
nearest thousandth of a gram using a microbalance. To minimize
the effect of water weight on mass determinations, worms were
placed on a preweighed square of 100-µm mesh Nitex cloth and
this mesh piece was blotted dry on a paper towel. We selected
wet weight because our worm supply was limited. Consequently,
we did not collect dry weights because this method is lethal and
would have depleted our worm supplies. It is likely that the
T. tubifex mean weights presented are high because of excess
water. However, a linear relationship between adult number and
total worm weight was observed (t = 23.5, P < 0.01, r2 =
0.96). This significant relationship indicates that, even though
the weights presented are probably high, the weight of excess
water increased linearly with worm number and therefore does
not bias our analyses. We combined adults and juveniles for
weighing because juveniles could not be weighed accurately
because water constituted a significant percentage (>90%) of
the mass when juveniles were weighed separately.
Feed type.—The objective of the first experiment was to
determine the optimal feed for T. tubifex. Three feed types
were tested: TetraColor tropical flakes (Tetra Holding Inc,
Blacksburg, Virginia), HBH Algae Grazers (HBH Pet Prod-
ucts, Springville, Utah), and cow manure. Algae Grazers con-
tained spirulina as the principal ingredient. Several manure “pat-
ties” were collected from a pasture in a nearby national forest.
The manure was homogenized and autoclaved prior to use. A
sample of manure was sent to the Utah State Department of
Agriculture and Food (Salt Lake City) for proximate analysis
(Table 1). One goal of this experiment was to measure the ability
of T. tubifex to assimilate the various feeds. Therefore, we pro-
vided an equal quantity of energy (calories) to each treatment.
We used a calorimeter to estimate the caloric content of each
 70 OPLINGER ET AL.
TABLE 1. Percentages of five ingredients in three feeds tested in the produc-
tion of Tubifex tubifex. The data on the cow manure were provided by the Utah
Department of Agriculture and Food. The information on the other two feeds
was taken from the packaging.
Algae
Ingredient Tetramin Grazer Cow manure
Crude fat 10.0 6.0 3.5
Crude fiber 2.0 6.0 17.3
Crude protein 49.0 28.0 6.6
Moisture 6.0 10.0 39.5
Ash 33.0 50.0 33.1
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feed. To do this, 0.5 g of feed was combusted, and the tem-
perature increase of 100 g of water was measured. The energy
content was calculated as: energy (J) = water mass (in grams)
× specific heat of water (in J/g) × change in temperature after
burning (in ◦ C). The energy content was then converted to kCal/g
of feed. The estimated energy densities did not vary among feed
types (one-way ANOVA: F 2, 28 = 0.983, P = 0.39). As a result,
an equal mass (8% body mass/d) of feed was provided to the
worms in each feed treatment. Eight replicate (four per each of
the two populations) beakers of 50 worms (13,372 worms/m2)
were provided each type of feed for 60 d. As a negative con-
trol, eight replicate beakers (four from each population) were
withheld feed for the same duration. Weekly, the water in the
beakers was exchanged and fresh feed was added. Beakers were
maintained at room temperature (15–19◦ C).
Ration.—The objective of this experiment was to determine
the feed ration (percent of body mass fed/d) that maximizes
T. tubifex growth and reproduction. This experiment was ini-
tiated after the feed type experiment and used the same adult
worms. Prior to initiation of the experiment, juvenile T. tubifex
were removed and adults were randomly reassigned to new
beakers. The mass of the adults introduced into each beaker
was determined. For this experiment, four ration levels were
tested: 0 (negative control, not fed), 2.5, 5.0, and 10.0% of
body mass/d. All worms were fed the same TetraColor flakes
as the previous experiment and were maintained at room tem-
perature (14–17◦ C). Water was exchanged and new food was
added weekly. This experiment lasted 60 d and eight replicate
groups (four per population) of 50 individuals (13,372/m2) were
exposed to each ration level.
Temperature.—The objective of this experiment was to de-
termine the effect of temperature on T. tubifex growth and re-
production. Temperature was manipulated by water baths. The
baths were constructed using either 26-L plastic buckets (25
cm in diameter, 45 cm deep) or rectangular, 45-L coolers (60
cm × 38 cm × 35 cm deep). The buckets were wrapped with
fiberglass insulation. Lids were constructed for both the buckets
and coolers using 2.5 cm thick foam insulation. Holes were cut
in the foam insulation lids to accommodate the same 400-mL
beakers used in the previous experiments. Each bucket or cooler
was designed to hold four beakers (two per population). The
buckets and coolers were filled with enough water to surround
the beakers containing T. tubifex. The water level, however, was
kept 5–10 mm below the top of the beakers. Small, submersible
aquarium pumps were used to provide circulation to the bathes.
Six temperature treatments were tested: 12, 15, 18, 21, 24, and
27◦ C. The 12◦ C and 15◦ C treatments were conducted in coolers
placed in separate rooms (one warmer, one cooler) of a relatively
cool building. During the last 30 d of the experiment, the rooms
began to warm, and the 12◦ C and 15◦ C beakers were moved
to separate temperature controlled refrigeration units. Tempera-
ture in the 18, 21, and 24◦ C treatments were manipulated using
aquarium heaters (Jaëger Model 3607; 200 W). These tempera-
tures were created in buckets, with two buckets per temperature
treatment. Finally, temperature in the 27◦ C treatment was ma-
nipulated in a cooler that was modified to accommodate a VMR
Model 1104 heat pump. Temperature in each cooler or bucket
was checked daily and adjusted as necessary. In addition, tem-
perature loggers (Hobo Pendant Model UA-001-XX or Hobo
Water Temp Pro Model H20–001) were used to record the tem-
perature of the buckets or coolers hourly. Aquarium air pumps
were used to aerate the beakers via air stones.
This experiment was started immediately after the ration ex-
periment and utilized the juveniles produced during the previous
experiment. Worms were added to the beakers at a density of
133,725/m2 (500/beaker). Eight replicate beakers (four from
each population) were tested at each temperature. Worms were
fed TetraColor flakes at 5% of body mass per day. As in the
previous experiments, water was exchanged and additional feed
was added weekly. This experiment lasted 120 d.
Rearing density.—The objective of this experiment was to
determine how stocking density influences the growth and re-
production of T. tubifex. Only individuals from the White River
source (mix of adults and juveniles) were used in this experi-
ment. Seven densities were tested: 2,675, 6,686, 13,373, 26,745,
66,863, 133,726, and 267,451/m2 (10, 25, 50, 100, 250, 500, and
1,000/beaker). Four replicate beakers of each density were es-
tablished. The beakers were held at room temperature (14–17◦ C)
and worms were fed TetraColor flakes at 5% of body mass per
day. This experiment was started immediately after the temper-
ature experiment, and it was assumed that the mean mass of
T. tubifex used in this experiment was identical to the mass mea-
sured at the end of the temperature experiment. This experiment
also lasted 120 d.
Statistical analyses.—Two measures of production were cal-
culated: (1) the average number of juvenile T. tubifex produced
per adult (number of juveniles recovered/number of adults re-
covered) and (2) the per-day increase in mass per milligram of
T. tubifex initially stocked. Analysis of variance (ANOVA) was
used to compare these production metrics. Preliminary analyses
showed no differences in production, growth, and recruitment
between source populations (all P > 0.16). Therefore, the data
from both populations was pooled for analysis. To meet the
assumption of normality, the data were loge transformed prior
 CULTURE OF Tubifex tubifex 71

TABLE 2. Results of four production experiments with T. tubifex that tested the effect of feed type, food ration, temperature, and initial adult density. Standard
errors are shown in parentheses. Different letters within columns and treatments indicate satistical differences (P ≤ 0.05) in least-significant-difference tests.

Number of
Experiment and Combined total Juveniles produced Daily mass produced
treatment Adults Juveniles weight (g) per adult (number) (mg/mg stocked)
Feed type
Tetramin 50.0 (0.8) z 945.0 (131.2) z 0.95 (0.03) z 18.9 (2.5) z 0.14 (0.01) z
Algae wafer 45.6 (5.4) z 683.6 (152.4) z 0.60 (0.09) y 16.2 (3.5) z 0.09 (0.00) y
Manure 30.9 (5.6) y 108.5 (17.5) y 0.23 (0.15) x 3.9 (0.5) y 0.02 (0.00) x
Control 49.6 (1.9) z 128.0 (35.3) y 0.22 (0.03) x 2.6 (0.7) y 0.02 (0.00) x
Ration
0.00% 49.4 (0.8) z 90.6 (31.0) y 0.27 (0.02) x 1.8 (0.6) y 0.00 (0.00) x
2.50% 50.5 (1.4) z 1,011.4 (166.3) z 0.54 (0.07) y 19.9 (3.1) z 0.03 (0.00) y
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5.00% 53.4 (2.2) z 1,737.4 (169.3) z 0.82 (0.12) z 32.7 (3.2) z 0.06 (0.00) z
10.00% 52.8 (2.3) z 1,668.9 (315.6) z 0.92 (0.16) z 30.9 (4.6) z 0.07 (0.01) z
Temperature
12◦ C 166.0 (38.9) x 2,129 (438) x 2.2 (0.5) z 18.5 (4.5) zy 0.19 (0.05) z
15◦ C 222.0 (34.2) yx 2,864 (651) yx 1.7 (0.3) z 17.9 (7.9) zy 0.15 (0.03) z
18◦ C 288.5 (33.3) zy 7,457 (1,528) z 1.9 (0.5) z 27.7 (5.9) z 0.17 (0.04) z
21◦ C 321.0 (45.2) zy 5,876 (1,320) zy 1.9 (0.5) z 18.6 (2.9) zy 0.17 (0.04) z
24◦ C 397.5 (74.4) xz 2,541 (587) x 1.7 (0.4) z 6.9 (1.5) y 0.15 (0.04) z
27◦ C 344.5 (96.0) zy 2,551 (759) x 1.6 (0.5) z 10.8 (3.9) y 0.14 (0.04) z
Density
2,674/m2 7.8 (0.9) t 213.5 (27.5) x 0.06 (0.01) x 28.6 (5.4) z 0.0080 (0.00) z
6,686/m2 19.5 (1.9) u 319.3 (70.2) yx 0.09 (0.01) yx 17.4 (4.5) zy 0.0078 (0.00) zy
13,372/m2 32.0 (2.3) v 227.3 (19.2) x 0.10 (0.01) zy 7.1 (0.3) y 0.0074 (0.00) yx
26,744/m2 66.3 (2.7) w 399.8 (90.7) yx 0.42 (0.22) zy 6.1 (1.4) y 0.0076 (0.00) zyx
66,863/m2 156.3 (17.6) x 647.3 (166.3) yx 0.48 (0.04) zy 4.3 (1.2) y 0.0074 (0.00) yx
133,720/m2 313.5 (37.5) y 2,773.5 (640.1) z 0.88 (0.09) zy 9.2 (2.0) y 0.0072 (0.00) x
267,440/m2 523.3 (49.4) z 1,742.0 (1,173.7) zy 1.85 (0.08) z 3.4 (2.3) y 0.0073 (0.00) yx

to analysis. We were not able to normalize the total mass and
production variables for the density experiment using common
transformations. Consequently, these data were analyzed using
a nonparametric Kruskal–Wallis H-test. All analyses were per-
formed using SAS version 8.0 (SAS 1998). Statistically signif-
icant model effects (α = 0.05) were investigated by comparing
least significant differences among all treatment combinations.
These comparison tests were performed using LSmeans state-
ments in SAS (SAS 1998).
RESULTS
Feed Type
The mass of T. tubifex placed in the beakers at the start of the
experiment did not vary among feed types (F 3, 27 = 0.39, P =
0.76). At the end of the experiment, the number of adults and ju-
veniles recovered and the combined and individual total mass of
adults and juveniles varied among feed types (all P < 0.01). The
number of adults recovered in the manure treatment was statis-
tically lower than in any of the remaining treatments (Table 2;
all P ≤ 0.04). No difference in the number of adults was ob-
served among the algae wafer, control, and Tetramin treatments
(all P ≥ 0.38). The number of juveniles and the total mass of
T. tubifex recovered was lowest in—but did not significantly
differ between—the control and manure treatments (Table 2;
both P ≥ 0.69). The number of juveniles recovered was greatest
for the algae wafer and Tetramin treatments and did not sig-
nificantly differ between the two treatments (T 1, 3 = 1.37, P =
0.18). In contrast, the greatest total worm mass was recovered in
the Tetramin treatment. Significant differences were observed
among feed types when both production variables were evalu-
ated (juveniles produced per adult and daily gain in mass/initial
mass; both P < 0.01; Table 2). For both variables, produc-
tion was lowest and did not differ (both P ≥ 0.06) between
manure and control treatments. Production measured as juve-
niles produced per adult did not differ between the Tetramin
and algae wafer treatments, but when measured as mass gain,
production was greater for the Tetramin than algae wafers
groups.
Ration
As in the last experiment, the initial adult mass of T. tubifex
did not differ among rations (F 3, 27 = 0.54, P = 0.66). Ration
 72 OPLINGER ET AL.
had no influence on the number of adults recovered at the end
of the study (F 3, 27 = 1.11, P = 0.36). The number of juveniles
recovered, however, did vary with ration (F 3, 27 = 46.50, P <
0.01). Juvenile numbers were lowest at the 0% ration (Table 2;
all P ≤ 0.01) but did not differ among the other rations tested (all
P > 0.11). The total mass of T. tubifex recovered also varied with
ration (F 3, 27 = 34.19, P < 0.01). The total mass recovered was
lowest at the 0% ration (Table 2; all P ≤ 0.04). The second
lowest mass was recovered at the 2.5% ration, and the total
mass recovered was greatest and did not differ between the
5.0% and 10.0% rations (Table 2; T 1, 3 = 0.41, P = 0.68).
Significant differences in production were observed among
rations (Table 2; both P < 0.01). When measured as the number
of juveniles produced per adult stocked, production was greatest
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for—but did not differ among—the 2.5, 5, and 10% body mass/d
rations (all P > 0.11); when measured as the daily increase in
mass per initial mass, production also was greatest for—and
did not vary significantly between—the 5% and 10% rations
(T 1, 3 = 0.20, P = 0.84). As before, production was lowest at
0%, followed by 2.5% (both P ≤ 0.01).
Temperature
Data from the temperature loggers showed that the temper-
atures achieved in our water bathes were similar to the desired
temperatures. For the 18, 21, 24, and 27◦ C treatments, achieved
temperatures were ±1.5◦ C from the desired in more than 98%
of the recordings. In no instances did the recorded temperatures
from one treatment reach the desired temperature of another
treatment (e.g., temperatures in the 18◦ C treatment were always
between 15.1◦ C and 20.9◦ C). The exceptions were the 12◦ C and
15◦ C treatments. In 5.1% of the recordings, temperatures in the
12◦ C treatment exceeded 15◦ C. Similarly, in 2.3% of the record-
ings, temperatures in the 15◦ C treatment were below 12◦ C. Still,
temperatures in the 12◦ C treatment were within 1.5◦ C of the de-
sired temperature in 88% of the recordings. Temperatures in the
15◦ C treatment were within 1.5◦ C of the desired temperature in
92% of the recordings. Despite this variation in temperature, we
successfully produced discrete temperature treatments.
As in the previous studies, initial adult T. tubifex mass did
not differ among temperature treatments (F 5, 36 = 0.42, P =
0.83). The survival of adults varied with temperature (F 5, 36 =
2.56, P = 0.04). Overall, survival at 24◦ C was greater than
at 12◦ C (T 1, 3 = 2.59, P = 0.01), but survival did not differ
among the remaining temperatures (Table 2; all P > 0.14).
Total juvenile recruitment also varied with temperature (F 5, 36 =
8.56, P < 0.01). Statistically, recruitment was greatest at 15◦ C
and 18◦ C but did not differ among the remaining temperature
treatments (Table 2; all P > 0.07). The total mass recovered
and the mass production (daily growth [mg] per stocked mass
[mg]) did not vary with temperature (both P ≥ 0.06). Significant
differences in the number of juveniles produced per adult were
observed among the temperatures (F 5, 42 = 3.16, P = 0.02).
No differences in juvenile production were observed among the
12, 15, 18, and 21◦ C treatments (all P > 0.15). A significantly
greater number of juveniles were produced per adult in the 18◦ C
and 21◦ C treatments than in the 24◦ C and 24◦ C treatments (all
P < 0.03).
Rearing Density
Since T. tubifex progeny from the temperature experiment
were randomly distributed among the beakers for the density
experiment, it was assumed that the initial mass did not differ
among treatments. We observed significant differences among
density treatments in the number of juveniles and adults and
total mass recovered at the end of the experiment (Table 2; all
P < 0.01). Significant differences in production were also found
(Table 2; both P < 0.01), indicating that density had an influ-
ence on per-capita performance. When measured as the average
number of juveniles produced per adult, production was greatest
for (all P < 0.01) but did not significantly vary between the two
lowest density treatments: 2,684 and 6,686/m2 (T 1, 7 = 3.80, P
= 0.15). Production was similar among all remaining treatments
(Table 2). When measured as the daily increase in mass per ini-
tial mass, production also varied significantly among treatments
(Table 2) and was greatest at the lowest density (2,674/m2; all
P ≤ 0.01). Production at this level, however, was statistically
similar to that at two other tested densities, 6,686 and 26,744/m2
(both P ≥ 0.13). Production was lower but similar at all other
densities (all P > 0.09). Production at these densities also did
not differ from production at 6,686 and 26,744/m2 (all P > 0.07).
DISCUSSION
Little information on the culture of T. tubifex is available in
the literature. Our study, however, presents a culture system that
is different from that of other T. tubifex studies in the literature,
most having used flow-through systems with substrates that con-
sisted of a mixture of cow manure and sand (Marian and Pandian
1984; Marian et al. 1989; Ahamed and Mollah 1992). Depend-
ing on the treatment combination, the production observed in
our experiments (0.36–52.0 mg/cm2) ranged from 0.08 to 11.5
times as great as that reported by Ahamed and Mollah (1992;
4.5 mg/cm2). These results indicate that the production system
that we describe is more efficient and can produce a larger mass
of T. tubifex more quickly than previously described systems.
An additional benefit of our system is that it can be set up in a
smaller space. Since our system is static, it requires less water
than other methods of T. tubifex culture. The setup we describe
can raise a large number of worms cheaply and with minimal
investment into care.
Most previous T. tubifex culture studies have utilized cow
manure as a primary food source (e.g., Marian and Pandian
1984; Ahamed and Mollah 1992). Other experimental studies
(not emphasizing culture), however, tend to rear T. tubifex on
a diet high in algal material (e.g., spirulina or Algamac 2000;
Kerans et al. 2005; Beauchamp et al. 2006; Rasmussen et al.
2008). In contrast to other culture studies, our results (Table 2)
show that the performance of T. tubifex fed cow manure is infe-
rior to those fed either Tetramin or algae wafers. In fact, in some
 CULTURE OF Tubifex tubifex
cases, worms fed cow manure performed worse than those in the
unfed control treatments. The composition (e.g., carbohydrate,
protein, lipid, etc.) of the cow manure used in previous studies
is not known. The nutritional composition of cow manure is
influenced by the diet of the bovine that produced the manure.
It is possible that the manure collected for our study was in
some way less nutritious than the manure used in other studies
or that perhaps some of the plants our cows foraged on released
chemicals that impair T. tubifex performance. The manure used
in our study was fresh. It is possible that T. tubifex are better
able to assimilate nutrients from less-than-fresh manure.
Our results demonstrate that T. tubifex are better able to uti-
lize nutrients assimilated from the Tetramin and algae wafers
than those from cow manure. The higher survival and produc-
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tion of T. tubifex in the algae wafer and Tetramin treatments
is probably due to the higher protein content of these feeds.
These findings seem to suggest that T. tubifex growth and re-
cruitment is optimized on a high protein diet. Feeds used in
fish culture typically contain 25–45% crude protein, higher pro-
tein levels leading to better fish growth (Tabachek 1986; Murai
1992; Webster et al. 1992). For fish, the source of protein and
its digestibility are important considerations since plant proteins
may lack certain amino acids and have lower digestibility co-
efficients (Alexis et al. 1985; Morales et al. 1994). In the wild,
T. tubifex appear to achieve the greatest abundance and perform
best in the presence of leaf litter (Lazim and Learner 1987;
Chauvet et al. 1993; Zendt and Bergersen 2000). Other studies,
however, have noted that wild distributions are more influenced
by the availability of microbes in organic debris (McMurtry
et al. 1983; Brinkhurst and Gelder 1991) than vegetation type.
It is likely that these microbes constitute a large percentage of
the diet of T. tubifex. The Tetramin decomposed faster than any
other food source tested (Oplinger, personal observation), and
it is possible that this quick decomposition provided a higher
abundance of microbe forage for T. tubifex than the other feeds
tested.
We found that ration had little effect on T. tubifex survival
and production. In general, these metrics were lowest at the 0%
ration, and statistically, no differences were observed among
the 2.5, 5, and 10% rations. Despite the lack of significance, all
of the metrics appeared to indicate that peak performance oc-
curred at the 5% ration. Little information on the effect of ration
on T. tubifex survival and production is available. In previous
T. tubifex culture studies, worms were provided an excess of
food (Marian and Pandian 1984; Marian et al. 1989; Ahamed
and Mollah 1992). Nonculture studies appear to have provided
T. tubifex rations similar to those used in our study (Kerans et
al. 2005; Beauchamp et al. 2006; Rasmussen et al. 2008).
Temperature is a factor that has received little attention in pre-
vious studies of T. tubifex culture. Ahamed and Mollah (1992)
mention that their T. tubifex were reared at 29◦ C. This is 2◦ C
greater than the warmest temperature that we tested. Noncul-
ture studies tend to rear T. tubifex at around 15◦ C. We observed
few differences in survival, growth, and production among the
73
temperatures tested. This suggests that as long as temperature
is between 12◦ C and 27◦ C temperature has a minimal effect
on growth and production. That is consistent with the notion
that T. tubifex are a cosmopolitan species adapted to a wide
range of environmental conditions (Zendt and Bergersen 2000).
Experiments have shown that domesticated cultures perform
well at a wide range of temperatures and that if culture oper-
ations are established using T. tubifex from that wild, efforts
should be made to rear these worms at temperatures that ap-
proximate those of the source where the T. tubifex were obtained
(Kerans et al. 2005).
We found that initial stocking density had a large influence
on production. Although net mass and reproduction increased
with density, average per-capita production (growth and repro-
duction) decreased with density. Studies of wild populations
have noted a similar density response (Bonacina et al. 1989a;
Bonacina et al. 1989b; Steinbach Elwell et al. 2006). The initial
stocking densities used in our study were considerably greater
than other T. tubifex culture studies (Marian and Pandian 1984;
Ahamed and Mollah 1992). It is not clear why the observed
density response occurred. The amount of forage provided was
scaled in relation to the initial stocking density. Therefore, it is
not likely that the observed density dependence was caused by
forage limitation. Bonacina et al. (1989b) postulated that there
is strong intra-stage competition between adults and juveniles.
This competition may be enhanced at higher densities. Stein-
bach Elwell et al. (2006) noted a reduction in reproduction at
higher densities. The reduced juvenile and mass production ob-
served in our study appears to corroborate the results of both
Bonacina et al. (1989b) and Steinbach Elwell et al. (2006).
Water quality can become a major concern when culturing
aquatic organisms in a static system (Piper et al. 1982). How-
ever, T. tubifex is a cosmopolitan species that appears to be more
tolerant of poor water quality than many other organisms (Aston
1973). Previous research has demonstrated that T. tubifex can
tolerate anoxic conditions (7.5–10% air saturation; Birtwell and
Arthur 1980), low pH (<3.5; Chapman et al. 1982), and pro-
longed exposure to ammonia concentrations exceeding 4 mg/L
(Aston 1973). We did not record water quality parameters during
our experiments, but based on these other studies it is not likely
that the performance of our cultures was inhibited by poor water
conditions. Furthermore, we did not observe extensive mortality
in any of our treatments. By aerating the beakers, the dissolved
oxygen levels experienced by our T. tubifex were probably close
to saturation. Also, the weekly water exchanges probably helped
keep concentrations of nitrogenous wastes below levels that are
considered toxic to T. tubifex. Finally, even at high rearing den-
sities and rations, the quantity of food we provided was small
(always <0.1 g of feed per beaker daily). This mass of feed was
probably not great enough to significantly degrade water quality
during our experiment.
Ecological differences among T. tubifex strains are poorly
understood. However, it is known that microhabitat preferences
can differ among strains (DuBey and Caldwell 2004). We used
 74 OPLINGER ET AL.
T. tubifex from two populations, one was entirely of the type
III lineage, whereas the other was predominately of the type
VI lineage (14% of individuals were type III lineage). Regard-
less, the fact that microhabitat preference differences are known
among strains (DuBey and Caldwell 2004) suggests that opti-
mal culture conditions found in this study are the same for all
strains. The research presented here helps identify methods that
can be used to mass-culture T. tubifex. Based on our findings,
four recommendations can be made regarding the culture of this
species. First, based on the diet test, Tetramin or a diet high in
algal material appears to support the best survival, growth, and
recruitment. Second, although T. tubifex appear to perform best
at rations of 5–10% of body mass/d, we recommend the 5% ra-
tion because this ration reduces food cost, waste, and potential
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water quality problems (due to rotting food). Third, tempera-
tures within the 12–27◦ C range appear to have little influence
on T. tubifex growth and survival, but recruitment appears to de-
crease at temperatures above 21◦ C. Fourth, although T. tubifex
fecundity is density dependant and decreases at concentrations
exceeding 2,674/m2, we believe that the optimal density de-
pends on the culture scenario. If space is limited, a substantial
number of juveniles can be reared if adults are stocked at a high
density. If space is not limited, then the greatest total juvenile
production is achieved at lower stocking densities.
ACKNOWLEDGMENTS
We thank B. Nehring and the Colorado Division of Wildlife
for providing the T. tubifex cultures used in this study. This
manuscript was greatly improved through comments by C.
Kohler and two anonymous reviewers. Funding for this research
was provided by the Federal Aid in Sport Fish Restoration Pro-
gram, project F-96-R, and the Utah Division of Wildlife Re-
sources.
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