THE ACTIVITY OF CHTTOBIASE IN THE MEDIUM: A BIOCHEMICAL ESTMATE OF DEVELOPMENT RATE EN PLANKTONIC CRUSTACEA A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by AKASH RENE SASTRI In partial fulfilment of requirements for the degree of Master of Science April, 2001 O Akash R. Sastri, 200 1
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THE ACTIVITY OF CHTTOBIASE IN THE MEDIUM: A BIOCHEMICAL ESTMATE OF
DEVELOPMENT RATE EN PLANKTONIC CRUSTACEA
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
AKASH RENE SASTRI
In partial fulfilment of requirements
for the degree of
Master of Science
April, 2001
O Akash R. Sastri, 200 1
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ABSTRACT
THE ACTIVITY OF CHITOBIASE IN THE MEDIUM: A BIOCHEMICAL
ESTIMATE OF DEVELOPMENT RATE IN PLANKTOMC CRUSTACEA
Akash R. Sastri
University of Guelph, 2001
Advisor:
Professor J-C- Roff
The activity of the molting enzyme chitobiase in the medium surrounding individuals and
populations of planktonic crustacea was investigated. Two applications of this enzyme
assay are presented as methods of estimating development rates in crustacean
zooplankton.
The correspondence between elevated chitobiase activity in the medium and the presence
of exuviae was confirmed in seven fieshwater cladoceran, and one fieshwater and six
marine copepod species. This biochemical cue of the molting event was applied as a
method of estimating the proportion of animals molting in a defined period of time
(development time). An estimate of Daphnia magna (2,000-2,100 p m size class)
development time was in close agreement with that derived by conventional incubations
(70.3 versus 75.1 hours respectively).
Chitobiase activity in the medium was found to Vary with body length in six tteshwater
cladocerans and six marine copepod species. Although the dopes of species specific
regressions differed, a significant common relationship was found (loglo [chitobiase
activity] = -1.19 + 0.89 loglo [body length], r2 = 0.79, p<O.0001). Under steady state
conditions in laboratory cdtures, the rate of decay of chitobiase in the medium was
balanced by its rate of production by molting animais. The rate of decay of the enzyme in
the absence of animais was therefore aIso its rate of production, which is a measure of the
average rate of development of the crustacean zooplankton community. Development
times for a Daphnia magna culture (2 determinations) and a Ceriodaphnia sp. - D.
magna rnixed culture were 65.4 vs. 62 hours, 59 vs. 67 hours and 46.6 vs, 50 hours, as
measured by this application of the chitobiase assay versus conventional molt rate
determinations respectiveIy.
ACKNOWLEDGEMENTS
During the last year of my undergraduate studies, I was exposed to some aspects of aquatic science research that both excited and compelled me to leam more. My sincerest gratitude to my advisor John C. Roff, for facilitating a learning expenence that has far exceeded any possible expectations I might have had when 1 began two years back. I thank John for his guidance, encouragement and infinite patience.
1 wodd also like to thank my advisory cornmittee members, Professor D.H. Lynn, and Professor J.S. Ballantyne for their help with d l manner of questions and carefûl review of my thesis. Some of my field work was conducted in Dorset, Ontario, where Professor N.D. Yan was very kind to provide me facilities, and his thoughtful insights were helpfùl and greatly appreciated.
My lab mates, Warren Cume, Susan Evans, Kem Finlay, Kim Rose and Richard Janutka were together responsible for creating a daily experience in the lab that was always exciting, thought provoking, and above d l fun to participate in, thank you.
I would also like to acknowledge the Department and fellow graduate students, specifically Ken Oakes and the boys in the Ballantyne lab for their fi-iendship and help in the lab. Also my ankle is as good as new, nearly, thanks to the kind efforts of Colin Darling, Michelle Campbell, Susan Evans and James Kowaleski, Cheers!
And lastly, and by no means least, my parents and sister have supported my endeavors in every possible way, for their love and support, 1 am always grateful.
TABLE OF CONTENTS
. ............................................... Chapter 1: Thesis background and rationale Pg 1
1 o d u c o n ................................................................................ Pg- 2
Figure 1. I . Successive changes in a crustacean integument during the molt cycle (after Drach 1939; Roff et aI. 1994)
Where stage D O to D 1 represents separation of epidermis fiom exoskeIeton and onset of apolysis; D 2 to D 4, digestion of old postecdysial layer and synthesis of new epicuticle and preecdysial Iayers; ecdysis, molting of old exoskeleton and liberation of molting fluid into the aqueous environment; A-B, de novo synthesis of new postecdysiai Iayer and; C, hIIy synthesized exoskeleton,
Chapter 2: Chitobiase assay for determining development time in
Crustacea
A.R. Sastri and J. C. Roff
To be submitted in Note format to HydPobiologia
Abstract
The proper calculation of secondary production in crustacean zooplankton depends on the
measurement of their growth rates. This in turn requires knowledge of development times and
molting rates. Detexmination of molt rates currently requires prolonged incubations of
individuals or batches of animals, which usually depends on finding the cast exoskeleton
(exuvia), We have found that chitobiase (one of two chitinolytic enzymes), which is released into
the medium at ecdysis (time of molting), serves as a simple and highly accurate method of
determining the proportion of animais molting during a time interval. Presence and activity of
chitobiase is rapidly and easily measured fluorometrically by release of methlyumbelliferone
f?om Methlyumbelliferyl-N-acetyl-glucosamine. The assay requires a single substrate and a short
incubation period of the water in which an animal has resided. Using cultures and natural
populations of fieshwater zooplankton, we determined the validity of this method by establishing
three criteria. 1) Planktonic Crustacea liberate the enzyme chitobiase at molt, as evidenced by
elevated chitobiase activity in the medium surrounding moIted individuals (presence of exuviae)
relative to non-molted individuals. This was established for adults and neonates of Daphnin
magna, neonates of Daphnia pulex, adult Ceriodaphnia sp. and copepodites of freshwater
copepods. 2) Chitobiase activity was measured in individual anirnals as small as 244 pn in
length. 3) The enzyme activity is stable at room temperatures when filtered (0.2 prn).
Developrnent times were calculated from numbers of animals molting as indicated by the
proportion of animals showing elevated chitobiase activity. Developrnent time using the
chitobiase assay was in close agreement with that derived by conventional incubations (70.3
versus 75.1 hours respectively).This method is applicable to ail marine and fieshwater planktonic
crustacea, and eliminates the need for prolonged incubations of animals and the Iabonous
microscopie search for exuviae.
Introduction
Estimation of growth rates in aquatic secondary producers still relies entirely on
conventional techniques. Growth rate (g) is the product of development rate (lm) and the
growth increment. Thus, g = (Ln(W2/WI))iD. While the growth increment (W2/WI ) can be
readify detemiined where developmental stages or size classes are recognized, it is the
developrnent time that is difficuIt and laborious to measure. In the absence of discrete cohorts,
laboratory or field determinations of development time generally require prolonged incubations
of individuals or batches of animals.
To address this difficulty, a biochemical technique has been developed and tested to
rapidly screen dozens or hundreds of individual anirnals in order to determine the fiequency of
rnolting in crustacean zooplankton and thus derive the development time. The method obviates
the need for microscope work and the need to search for cast exuviae. It is based on the enzyme
chitobiase, one of two chitinolytic enzymes found in a diversity of organisms, including al1
crustacea-
Chitin is a simple polymer of P-(1-4) linked N-acetyl-glucosamine and is the primary
structural constituent of al1 arthropod exoskeletons. At apolysis (the start of premolt), the
exoskeleton separates fiom the epidermis. During premolt, the enzymes chitinase and chitobiase
catalyze a partial recycling of chitin from the old to the new exoskeleton. Several studies of
planktonic crustacea have observed increased chitobiase activity during premolt (Buccholz 1989,
Espie & Roff 1995). At the moment of and subsequent to molt (ecdysis), elevated chitobiase
activity can be measured in the medium surrounding the organisms (Vrba & Machacek 1994).
The chitobiase assay is therefore a potential index of actual growth rate (e.g. Espie & Roff 1995,
Oosterhuis et al. 2000).
In another study, we (Sastri and Roff 2000; Chapter 3) showed that the average
development tirne of crustacean zooplankton populations can be derived fiom a knowledge of
the size-specific rate of production of chitobiase and the turnover rate of this enzyme in the
medium. However, this method has not yet been applied to natural populations, and there may be
limitations to its application (see Chapter 4). For example: where development of populations is
not isochronal, where background levels of chitobiase are contributed fiom non-zooplankton
sources or where attention focuses on a particular species, the assay proposed by Sastri and Roff
(2000) may not be appropriate. The objective of the present study was to determine whedier the
activity of chitobiase in the medium following ecdysis could serve as a simple method of
determining the proportion of animals molting during a given time interval, fiom which the
development time cm be derived.
Methods and Results
Individuals were incubated in test tubes containing synthetic fieshwater (see Roff et al.
1994; Appendix 1) for 6 hrs at 22 OC. Volume of medium and enzyme-substrate incubation time
were adjusted depending on the size of animals (Table 2. L), but al1 assays were nin at saturating
substrate concentrations (Appendix 2). A synthetic freshwater was used in order to reduce any
background chitobiase activity. At the conclusion of incubations, the medium was exarnined for
the presence of exuviae, either by eye or under 20 X magnification. Aliquots of 0.7 ml of
medium were removed fiom tubes in which animals had molted, and chitobiase activity was
measured immediately. Chitabiase activity was measured as an increase in fluorescence with
time in 0.7 ml of incubation medium following the addition of 150 pl of 0.4 mm01 (final
concentration) methy lurnbellifery 1 -N-acetyl- p -D-glucosamhide (MUF-NAG) (Sigma C hernical
Co.). Concentrated substrate stock dissolved in Cellosolve (Sigma Chernical Co.) was diluted to
desired concentration in 0.15 M citrate phosphate bufTer, pH 5.5. Medium sample and substrate
were incubated for 10-40 min (see Table 2.1) and the reaction stopped with the addition of 150
p1 of 0.25 N NaOH. Immediately following the addition of NaOH, fluorescence of liberated
rnethylurnbelliferone (MUF) was measured at 360 nm excitation and 450 nm emission using a
Perkin Elmer LS50 Luminescence Spectrometer. Al1 assays were conducted at 22 OC. Controls
(synthetic freshwater) were run (in triplicate) to assess background fluorescence of the substrate
and any background activity associated with the incubation medium itself. Chitobiase activity is
expressed as nrnol MUF liberated per 1 O min (jbr conversion ofjzuorescence values ta chitabiase
activity see Appendices 3a-g).
The reIationship between molting and an elevated chitobiase activity was first examined
using neonates (600-640 pm) of the cladoceran Daphnia pzilex. An asynchronous Iaboratory
culture was used as the source of animals. Cultures were maintained under a 16L:8D hour
photopenod and were fed Scenedesmus sp., ad libitum. Neonates, released by adult females,
were each incubated in 1.5 ml of synthetic freshwater, and exarnined every three hours. The first
individuals to molt were removed and the rernaining individuds were inspected every hou . At
each subsequent interval (each hour), individuals were examined for presence of exuviae and a
0.7-ml aliquot of medium was removed for enzyme assay. We found that the presence of exuviae
was always tied to an elevated chitobiase activity relative to background levels (Table 2.1).
We also tested for the presence of released chitobiase in the medium surrounding other
zooplankton species. assay was applied to adults and neonates of Daphnia magna, neonates
of Ceriadaphnia sp., and copepodites of Diapromus sp. Again, a strict relationship was observed
between production of exuviae and an eIevated chitobiase activity in al1 trials (Table 2.1).
Oosterhuis et al. (2000) have dso used this assay on the marine copepod Temora longicornis.
Given the universality of chitin biochemistry, we believe that this assay is applicable to al1
crustacea.
In order to determine whether the chitobiase assay could measure the proportion of
animals molting, we compared development time calculated by direct observation against that
derived via this chitobiase method. A conventional measure of development time was
determined by incubating individual ( ~ 2 0 ) D. magna (2,000-2,200 pm) in 3 -0-4.0 ml of culture
medium. Each individual was examined every 3 to 6 hours for the presence of exuviae.
DeveIopment time was calculated as twice the mean time to observation of first m o k This value
was not significantly different fiom direct observation (n=10, mean=74.9 , t=-0.0 103, P=0.992)
of the entire intermolt perîod.
To deterrnine the proportion of animais molting using the chitobiase assay, we randomly
removed and incubated 30 anirnals (2,000-2,200 pm) in 3.0 ml of synthetic fieshwater. Animal
incubation periods Iasted 6 hours, at the conclusion of which, aliquots of 0.7 ml were removed
and chitobiase assayed as per above. Sixteen consecutive 6-hou incubations and enzyme assays
were performed on animals initially removed for each interval fiom the same culture vesse1 as
that used for our conventional determination. We randornly chose animals and contulued to
monitor rnolt rate in this manner over 96 hours in order to allow for any possible die1 periodicity
or synchronicity in time of molt in the population. Development times were calculated fiom
numbers of animais molting as indicated by the proportion of animals showing elevated
chitobiase activity. Development tirne using the chitobiase assay was in close agreement with
that derived by conventional incubations, 70.25 (n= 16, S.E.=9.18) versus 75.1 ( ~ 2 0 ,
S.E.=5.75) hours respectively. The incubation medium used for al1 "chitobiase" determinations
of molt rate was synthetic freshwater (Appendix 1). Anba l s were therefore d e d during the
course of each 6 h o u incubation. Thus, we also compared the proportion of animals molting in 6
hour incubations while housed in medium supplemented with food or without ( 1 ~ 1 0 for each
incubation medium).
The duration of an incubation period is dependent, in part, on the length of time
chitobiase in the medium remains detectable at levels above background. Furthemore, a rnethod
which preserves chitobiase activity in samples for long periods may be useful when assessment
of activity is not irnrnediately possible. Thus, we examined the time course for decay of
chitobiase released into the medium following molt (Appendix 4) . Chitobiase activity was
assayed (n=5) at 0, 3,6, 12, and 18 hours. Mean activity remaining at 12 hours was 79.2% of that
measured at time zero (S.E.=5.22), and had declined at 18 hours to 49.5% (S.E.=8.63).
Accordingly, candidate methods to preserve sample activity were examined. These methods
included combinations of fieezing and chernical treatments. Sarnples were initially assayed at
room temperature and again following specific treatments. The best retention of activity, afier 7-
9 days storage at 5 OC , was 85.8% (mean n=10, S.E.= 0.13) of initial activity, following the
addition of 0.1 mm01 DTT (dithiothrietol) and storage at 5 OC. Other suitable treatments included
storage of 0.2 pm-filtered sarnples at -10 and +5 OC. It was found that filtering alone exerted a
major effect on the rate of chitobiase decay in solution. The activity in filtered sarnples (n=l2)
changed little in the fist 48 hours, after which activity decreased but remained consistently
above 60% after 10 days (Appendix 5,6).
Cautions, Optimization and Application
In adult D. magna individuals (2,000-2,200 p), elevated chitobiase activities were
aiways observed in the presence of exuviae. Likewise, the same relationship was observed with
D. pulex neonates (600-640 p), but in one particular tnal, of 10 of 32 individuals molting over
6 hours, chitobiase activity was detected in the medium 1 hour or less before the observation of
exuviae. Vrba & Macacek (1 994) observed a similar phenornenon with Daphnia puliearia. Thus,
if molting rate is assessed simply fiom the presence of chitobiase, an introduction of error (-5%)
might occur. This error was calculated as the product of the proportion of animals molting with
chitobiase activity before evidence of molt (exuviae) to the total number molting (l0/32) and the
reciprocal of the duration of the experiment (1/6). Thus, molt rates cdculated using the
chitobiase method would tend to overestimate the number of animds molting and underestimate
development time compared to methods dependant on an observation of exuviae.
At the conclusion of incubations, care should be taken to confim that no individuals have
died. We found varying degrees of chitobiase activity in the medium surrounding dead
individuals. Variation may depend upon the length of time an individual has been dead, and upon
the extent to which enzyme is liberated from epidermal vesicles or else the digestive tract.
Furthermore, apolytic individuals that die hold the potential to release more enzyme into the
medium following death.
Optimal assessment of chitobiase activity relies on both the nature of the incubation
medium itself and the length of incubation period. If not 0.2 pm-filtered or autoclaved, native
medium rnay also be contaminated with bacterial, flagellate, andior crustacean chitobiase. Thus,
discriminating between background and chitobiase released by smaller individuals may be
difficult. A simple increase in the Iength of enzyme-substrate incubation period may alleviate
d i s , but prolonged enzyme reactions may result in substrate limitation. Thus, in order to
optimize differences in fluorescence between controls and treatments we used pre-filtered
synthetic freshwater or seawater.
However, the use of filtered water precludes feeding during the incubation period,
perhaps artificiaIly proIonging the intermolt period and causing an overestimate of molt period
duration. Thus, the Iength of incubation period was also of import. We found no bias in molt
ratio between animals incubated in synthetic and unfiltered culture medium (t-test, t = -0.234,
P=0.8 18). Regardless of food availability, an apolytic animal is committed to molt (Shreeve et al.
1998). Thus, the length of incubation periods shodd not exceed the duration of the apolytic
phase (i.e.clO% of molt cycle for D. magna reared at 22 OC). Furthemore, the length of an
incubation period is aiso limited by the rate of decay of liberated chitobiase, which is enhanced
by the presence of microorganisrns in the medium (Vrba & Macacek 1994; Oosterhuis et al.
2000; Sastri & Roff 2000). Bacteria may be introduced to the incubation medium, either attached
to the animals' exoskeleton or released fiom the digestive tract. Depending on the species, the
presence of bacteria may also introduce cell-bound chitobiase activity into the incubation
medium. Thus, the longer the incubation period, the greater the potential for an increase in
"background" chitobiase activity. Chitobiase activity assayed in the medium surrounding non-
molted individuals (background) was observed to increase with duration of incubation (Figure
2.1). Therefore, an experimental protocol (duration of incubation etc.) will be a balance between
the duration of apolysis, the total amount of chitobiase produced (crustacean and cell-bound),
and its rate of degradation before assay. The optimd duration for individual D. magna incubated
at 22 "C was found to be 6 hours or less (Figure 2.1).
The incubation protocol should be based on three factors: 1) degree of synchrony or
asynchrony in a population; 2) the rate of degradation of released chitobiase, which is a function
of both temperature and total microbial activity; and the 3) expected duration of the molt cycle (a
function of temperature, food availability and body size (see Vidal 1980, Huntley and Lopez
1992, Hopcrofi and Roff 1995). In an asynchronous population, 30 single animals were
incubated for 6 hours when the expected duration of the moIt cycle was approximately 60 hours.
Thus, during this 6-hou period one wodd expect 3 animals to molt and show chitobiase activity
in the medium. Clearly the more replicates run, the greater the accuracy of the estimate of
development tirne. Many natural populations of planktonic crustacea show some degree of
synchrony in development, including strong die1 patterns of molting (Hopcrofi et al- 1998). This
should be considered in determining population development times. Continuous replicates (as
discussed above) of incubations throughout the course of the expected molt duration will reved
the existence of such die1 cycles.
Sample volumes and incubation times for enzyme-substrate reactions depend on body
size since the amount of enzyme released is a function of body size within a species (Vrba &
Machacek 1994; Oosterhuis et al. 2000). This relationship appears to extend across a number of
species (Fig 2. l), and c m potentially be used as a measure of molt rate in whole crustacean
communities (Sastri and Roff 3000). A divergence between chitobiase activity and background
activity with increased substrate-incubation time was observed (Appendix 2). Thus, greater
sensitivity can be obtained by prolonged enzyme-substrate reaction time. The smallest animals
that were assayed in this manner were Ceriodaphnia sp. neonates (244-304 pm).
This method depends only on detection of the enzyme's activity in sampIes (at a level
significantly above background), not on a measure of the actual rate of reaction. Provided the
incubation and enzyme reaction conditions are optimal (as discussed above), 50% or less of
remaining enzyme activity (relative to the initial reading) will be sufficient to discriminate
molted fkom non-molted individuds. Thus, we investigated the rate of decay of chitobiase
released into the medium and several methods of rnaintaining enzyme lability.
In surnmary, we have shown that the presence of chitobiase in the medium following
ecdysis is a simple surrogate index of molt and hence development time. Development tirne is a
fùnction of temperature, body size, and food concentration (Vidal 1980). The chitobiase method
can be used under any combination of these variables because it is simply an index of the
fiequency of molting animals. Aithough exuviae in Daphnia can be easily seen, those of its
neonates and the smaller stages of copepods are more difficult to find, or may be consumed
following molt. For these reasons, we suspect that there may be substantial biases in some
estimates of molt rates in smaller crustacea. Our method ailows rapid screening of large numbers
of animals and does not require specific calibration. Further, chitobiase activity remains stable
after filtration such that sarnples can be maintained for future deterrnination. The assay is highly
sensitive and can be used on single microcrustacea as small as 244 pm from the piankton and
benthos of both fieshwater and marine environrnents.
References
Buccholz, F. 1989. Molt cycle and seasonal activities of chitinolytic enzymes in the
integument and digestive tract of the Antarctic krill, Euphausia superba. Polar Biol.,
9:3 11-3 17
Espie, P.J., and Roff, J-C. 1995. A biochemical index of duration of the molt cycle for planktonic
Crustacea based on the chitin degrading enzyme, chitobiase. Lirnnol. Oceanogr.,
40: 1028-1034
Hopcroft, R.R., and Roff, J-C. 1995. Zooplankton growth rates: extraordinary production by the
larvacean Oikopleura dioca in tropical waters. J. Plankton Res., 1 7:205-220
HopcrofS R. R., Roff, J. C., Webber, M. K., and Witt, J.D.S. 1998. Zooplankton growth rates:
influence of size and resources in tropical marine copepodites. Mar. Biol., 132: 67-77.
Huntley, M.E., and Lopez, M.D.G. 1992. Temperature-dependant production of marine
copepods: a global synthesis. Amerïcan Naturalist., l4O:2O 1-242
Oosterhuis, S.S., Baas, A.B., and Klein Breteler, W.C.M. 2000. Release of the enzyme
chitobiase by the copepod Ternoru [ongicornis: characteristics and potential tool for
estimating crustacean biomass production in the sea. Mar. Ecol. Prog. Ser., 196: 195-206
Roff, J-C., Kroetsch, J.T., and Clarke, A.J. 1994. A radiochernical method for secondary
production in planktonic cnistacea based on the rate of chitin synthesis. J-Plankton Res-,
l6:96 1-976
Sastri, A.R., and Roff, J-C. 2000. Rate of chitobiase degradation as a measure of development
rate in planktonic crustacea. Can. J. Fish. Aquat. Sci., 57: 1965-1968
Shreeve, R.R., Ward, P., and Murray, A.W.A. 1998. Moulting rates of Calanzis helgolandiczts:
an intercornparison of experimental methods. J. Exp. Mar. Biol. Ecol-, 224: 145-1 54
Vidal, J. 1980. Physioecology of zooplankton. 1, II. Effects of phytopIankton concentration, an4
body size on the growth rate of Calanus pacificus and Pseudocalanus sp. Mar. Biol.,
56: 11 1-134
Vrba, J., and Machacek, J. 1994. ReIease of dissolved extracellular P-N-acetylglucosarninidase
during crustacean molting. Limnol. Oceanogr., 39:7 12-7 16
Table 2.1. Sizes, incubation volumes, and enzyme reaction times for chitobiase detenninations on various species of microcrustaceans-
Species Size (pm) Nurnbers Chitobiase Nurnbers Chitobiase Incubation Substrate not molting activity* molting activity* volume (ml) incubation time
(no exuviac) (exuviae) (min)
Daphnia magna 2700-2900 7 16.3 S.E.=0.825
Daphnia magna 840- 1 050 7 15.9 neonates S.E.=O.S I Daphnia p u k t 600-640 36 12.9 neonates S.E.=0.04 Ceriodaphnia sp. 244-304 17 16-4
S.E.=0.53 Diapromus sp. 600-630 17 17.3
S.E.=0.878
*Chitobiase activity expressed as nrnol methylumbelliferyl (MLTF) liberated during substrate incubation penod. Al1 activity values have been corrected to 3 .O ml incubation volume and for background fluorescence.
O 500 1 O00 1500 2000 2500 3 O00 3 500 4000
Body Iength (pm)
Fig. 2.1. Relationship between body size and released chitobiase activity in molted and non-
rnolting animals. O-Ceriodaphnia sp.; Ei - ~ a ~ h n i a plex; and A-Daphnia magna. Change in background activity after: -6 hours; and . - 12 hours, with non-molted animals.
Chapter 3: Rate of chitobiase degradation as a measure of development rate in planktonic Crustacea
A.R. Sastri and J. C. Roff
Published in October 2000 as a Rapid Communication in
Canadian Journal of Fisheries and Aquutic Sciences 57: 1965- 1968
Abstract:
We have developed a method to determine development tirne (molt rate) in both single and
mked populations of crustacean zooplankton, based on turnover of the chitïnolytic enzyme
chitobiase in the arnbient medium. We examined the relationship between body size and
chitobiase activity released into the medium following molt in three fieshwater cladoceran
where: [CB] = chitobiase activity; ni = # of anirnals Sn size cIass i; li = mean length of
animais in size class i; CBi = rate of production of chitobiase per animal in size class i; D =
average development time in days (between molts); NA = indicates aliquots fiom which animals
have been removed; c = indicates whole cultures. Note that -under steady state conditions:
(A[CB]/At)c = O.
Three separate assessments of development times were made using this technique, two on
D. magna monocultures and one on a mixed Ceriodaphnia sp. + D. magna culture. The rate of
decay of chitobiase in culture water without animais [(A[CB]/At)NA] was detennined as the total
change in activity over 24 hours, corrected for culture tank v-olume (Figure 3.2). Total chitobiase
production by the populations in each culture [((Cni.li.CBi)cD)] was calculated using the body
size to chitobiase activity regression (Figure 3. l), and animal abundances and size distributions
from three subsarnples (Appendix 7-8) of 250 rnL volume f iom each culture.
There was a close correspondence between development times estimated fiom chitobiase
turnover (eqn. 2) and those derived independently (fiom eqn- 1 ; see Appendix I O ) as follows:
65.4 vs 62 hours, 59 vs 67 hours, and 46.6 vs 50 hours, respectively.
Discussion
Chitobiase activity is known to be a function of body size within a species, both in whole
animal homogenates (Espie and Roff 1995a) and following release after molt (Oosterhuis et al.
2000). We have now shown that chitobiase activity is a function of body size arnong three
cladoceran species, and that a single mass-specific regression describes the relationship. Further,
the rate of decay of this enzyme in the medium can be used as a measure of the average rate of
deveIopment of the crustacean zooplankton community in cultures in the laboratory. The
technique should be applicable to al1 planktonic crustaceans, and to their natural populations in
the field. However, we do not yet know how well a single size-activity regression may describe
relationships in natural communities of zooplankton (see Runge and Roff 2000).
Detennining development tirne in this manner is attractive because of the ease with
which chitobiase is assayed; it is simple, inexpensive, and sensitive. We can measure the enzyme
activity released by a single animal of 244 pm in length. The method also eliminates repeated
handling and sacrifice of individuals, lengthy incubations, and laborious examination for
exuviae. A significant advantage of the method is that in situ water with natural food can be used
as the incubation medium. Our study was conducted at 22 OC. However the method c m be used
at any temperature. A valid calculation of development time requires only that chitobiase activity
in water samples and those used in the construction of chitobiase-body size relationships should
be at the sarne temperature. However, if this is not possible, values can be corrected for
temperature based on knowledge of the enzyme's temperature dependence (see Espie and Roff
1995b).
Chitobiase activity is found naturally in many bodies of water (Vrba and Machacek
1994). Individual animals were therefore incubated in synthetic fieshwater without food in order
to reduce background readings. Note that this does net introduce a bias into the method because
unfed individual animais are only used to establish the size-activity relationship, net to determine
the molt rate itself. Ideally, the incubation periods should be kept to a minimum, in order to
avoid significant decay of the enzyme. Some enzyme activity was detected in non-molting
animds. This is likely due to chitobiase released fiom the digestive tract. Although these values
were low (< 10% of activity in molting animals), we are now exploring the relationship between
the length of incubation period and the background activity.
It should be clearly noted that this method derives an average size-weighted development
time for a population of animais in a given body of water (see eqn. 2 above). The assurnption is
therefore that development times of al1 animais within a container are very similar (referred to as
'isochronality' for developmental stages within a species). However, developrnent is not
isochronal within the cladocera (see eqn. 1 above) or for copepods living under food limited
conditions (e-g. Hopcroft et al. 1998). However, when animals of a restricted size range
dominate the containers, the development times should be accurate and appropriate. Therefore,
this method is potentially applicable to open waters, lakes, and oceans where the size range of
crustacean zooplankton is restricted, or where populations are developing at nearly isochronal
rates.
Most natural water bodies are divided into upper and lower therrnal Iayers containing
smaller and larger crustacean zooplankton respectively. Therefore, it may be possible to apply
this method withh such compartments. Indeed, various strategies might be employed, such as
incubating individuals of a specific size fi-action (e.g. an artificial cohort, see Hopcroft et al.
1998) in rnicrocosms. Then, once the chitobiase activity versus body-size relationship is
developed for the zooplankton of a given region, only the size spectnim and abundance of
animals need be known and the rate of decay of enzyme measured to derive development times.
References
Azarn, F., Fenchel, T., Field, J.G., Gray, J.S., Meyer-Reil, L.A., and Thingstad, F. 1983. The
ecological role of water column microbes in the sea. Mar. EcoI. Prog. Ser., 10: 257-263.
Berges, J. A., and Bailantyne, J. S. 1990. Size-scaiing of whole body maximal enzyme activities
in aquatic crustaceans. Cam J. Fish. Aquat. Sci., 48: 2385-2394.
Conover, R.J., and Francis, V- 1973. The use of radioactive isotopes to measure the transfer of
materials in aquatic food chains. Mar. Biol., 18: 272-283
Espie, P.J., and RoE, J-C. 1995a. A biochemical index of duration of the molt cycle for
planktonic Crustacea based on the chitin degrading enzyme, chitobiase. Limnol.
Oceanogr., 40: 1028-1034.
Espie, P.J., and Roff, J-C. 1995b. Characterization of chitobiase fkom Daphnia magna and its
relation to chitin flux. Physiol. Zool., 68: 727-748.
Hopcrofi, R. R., Roff, J. C., Webber, M. K., and Witt, J.D.S. 1998. Zooplankton growth rates:
influence of size and resources in tropical marine copepodites. Mar. Biol., 132: 67-77.
Oosterhuis, S.S., Baars, M.A., and Klein Breteler, W.C.M. 2000. Release of the enzyme
chitobiase by the copepod Temora longicomis: characteristcs and potential tool for
estimating crustacean biomass production in the sea. Mar. Ecol. Prog. Ser., 196: 1 95-206.
Roff, J-C., Kroetsch, J.T., and Clarke, A.J. 1994. A radiochernical method for secondary
production in planktonic crustacea based on the rate of chitin synthesis. J. Plankton Res,.
16: 961-976.
Runge, J. A., and Roff, J.C. 2000. The measurement of growth and reproductive rates. Ch. 9. In
ICES Zooplankton Methodology Manual. Edited by R.P. Harris, P.H. Wiebe, J. Lenz,
H.R. Skjoldal, and M. Huntley. pp. 401-454.
Steeman-Nielsen, E. 1952. The use of radio-active (c") for measuring organic production in the
sea. J. Cons. Int. Explor. Mer., 18: 11 7-140.
Vrba, J., and Machacek, J. 1994. Release of dissolved extracellular P-N-acetylglucosarninidase
during crustacean molting. LimnoI. Oceanogr., 3 9: 7 12-71 6.
Fig. 3.1. The relationship between chitobiase activity released by individual animals after
molting, and body size in: Ceriodaphnia sp. (O), Daphnia pulex (BI), and Daphnia magna (A) (logio[activity] = -1.75 + 1 .O7 logi&ize], r2 =0.82, p< 0.0001). Each specie's specific regression was significantly different (pc0.05).
Time (hours)
Fig. 3 -2. Change of chitobiase activity in whole cultures of cladocerans: A mixed Ceriodaphnia sp. and Daphnia magna, a Daphnia magna alone. Change of chitobiase activity in aliquots fkom cultures fkom which animals have been removed: A mixed Ceriodaphnia sp. and Daphnia magna, O and 17 Daphnia magna alone.
Chapter 4: Towards an in situ application of the free chitobiase assay for estimating development time in planktonic Crustacea
Abstract
The presence and rate of decay of the chitinolytic e-e, chitobiase, in the medium, has
been used to estimate development t h e in both single and rnixed laboratory populations of
freshwater cladocerans. As a prelirninary investigation into the viability of field applications of
th is approach, it was necessary to determine the extent to which the relationship between
chitobiase activity and body length was conserved arnong several species (both freshwater and
marine) of microcrustacean zooplankton. Samples of both fieshwater and seawater were also
examined in order to determine whether chitobiase (of crustacean origin) could be assayed, and
its rate of decay followed in the natural environment. A measurable rate of chitobiase decay was
observed in native fieshwater sarnples. Furthemore, chitobiase activity, attributed to molting
crustaceans was assayed in both native fieshwater and seawater samples, and distinguished fiom
ce11 bound sources (>0.2 pn). A significant overall regression of chitobiase activity on body size
was observed for 12 fieshwater and marine species; (loglo [chitobiase activity] = - 1.1 9 + 0.89
loglo [length], 2 = 0.79, p<0.000 1). Each of the species specific regressions, however, were
found to be significantly diffèrent from each other and the overai1 regression.
4. I Introduction
Two unique methods of estirnating development time in planktonic crustacea are
described in Chapters 2 and 3. Both methods are based on an assay of chitobiase liberated into
the aqueous environrnent by rnoltïng individuals. Development of these methods in the
laboratory suggest that they hold promise as routine measures of development rate in natural
comunities of planktonic crustacea. A valid in situ application of the chitobiase method (as per
Chapter 3) is dependant on the satisfaction of two conditions. First, estimates of development
rates for natural zooplankton communities (rnixed species populations), require a relationship
between fiee chitobiase activity and body size, that is applicable across species. This relationship
formed the ba i s of the laboratory trials (Chapter 3), but inclusion of other species is fundamental
towards its broader application. Thus, I explored free chitobiase activity in four additional
freshwater cladoceran species and six species of marine copepods. Secondly, chitobiase liberated
by planktonic crustacea must be detectable, and its rate of decay memurable, in the natural
environrnent. Thus, the activity of chitobiase in sarnples of both natural freshwater and marine
environrnents was investigated.
4.2. Methods
The activity of chitobiase liberated by four additional cladoceran species was exarnined
relative to body length. Sampres of HoZopedium gibberurn and Daphnia dubia were collected late
in Septernber, 2000 frorn Plastic and Dickie Lakes, Dorset, Ontario. Chitobiase activity was
assayed within 2 days of sampling. Daphnia galeata and Daphnia pulicariu were collected fiorn
Guelph Lake, Guelph, Ontario, and maintained in the laboratory (as per conditions discussed in
Chapters 2 and 3). Individuais were processed for chitobiase activity within 1 week of collection.
Marine copepods were sampled regularly fkom late July to mid-Ausst 2000 from
Passamaquoddy Bay, New Brunswick. Individuals were incubated within I day of collections.
Free chitobiase was assayed in individual incubations of 2.0-3.0 ml of synthetic
freshwater (Appendix 2 ) for the cladocerans D. galeara, D. dzrbia, D. pukaria, and H
gibberzm. Al1 assays were conducted at 22 OC as per reaction conditions described in Chapters 2
and 3. Marine copepods were individually incubated in 2-04 .O m i synthetic seawater (Crystal
Sea Brand) for 6-9 hours. At the conclusion of incubations, each sample was examined (under 20
X magnification) for the presence of exuviae. Two aliquots of 0.7 ml of incubation medium were
removed for chitobiase assay.
A11 enzyme-substrate reactions were carried out at 14 "C (corresponding to both in situ
and incubation temperatures) with 0.4 mm01 methylurnbelliferyl-N-acetyl-PD-glu cos^
( W - N A G ) dissolved in 0.15 M CPB (pH 5.5). Reactions were stopped after 20 minutes with
the addition of 0.25 N NaOH and I M EDTA. Vrba & Macacek (1 994) attributed cloudiness in
their final solutions to alkalization caused by the addition of NaOH. Clear solutions were only
obtained in seawater with the addition of EDTA. The rate of MUF Iiberation was determined as a
change in fluorescence read on a Turner Designs (TD 7000) fluororneter at 360 nrn excitation
and 450 nm emission. Parallel blanks (synthetic seawater) were nin in triplicate to determine any
background fluorescence. Fluorescence values were cross-calibrated with fluorescence read on a
Perkin Elmer Luminsecence Spectrometer used for al1 fi-eshwater studies (Figue 4.1). In order to
facilitate comparison to fieshwater species, chitobiase activity was corrected to 22 OC, as per the
temperature relationship described by Espie and Roff (1 995a). lmmediately following chitobiase
assays, body length was measured for al1 individuals. H. gibberum was measured as per Yan and
Mackie (1987), D-galeara, D. puliearia and D. dubia as per Chapters 2 and 3 (top of head to base
of tail spine) and the marine copepods length of cephalothorax (as per McLaren et al. 1988).
Aliquots of seawater (n=3 were obtained fiom Passamaquoddy Bay, New Brunswick. Samples
were fust passed through 60-pin mesh to remove any crustaceans. Chitobiase in both 0.2-pm
filtered and unfiltered fractions was assayed in order to distinguish crustacean fkom ceII bound
activity. The linearity of the chitobiase-MUF-NAG (0.4 m o l ) reaction was tested over the
course of 40 minutes in order to confll~n substrate saturation. ALI reactions were stopped with
the addition of 0.25 N NaOH and 1 mol 1-' EDTA.
The rate of decay of chitobiase in sarnples of fieshwater (n=3), collected fiom Plastic
Lake, Ontario was also examined. Samples were initially passed through 60 p.m mesh (to remove
any crustaceans) and the rate of decay monitored. Treatments were assayed three times over 24
hours. Both 0.2-pm filtered and unfiltered sub-sarnples of the unfiltered sarnple were assayed at
each tirne interval. Al1 chitobiase reactions were run with 0.4 mm01 MUF-NAG, for 10 minutes
and stopped with the addition of 0.25 N NaOH.
4.3 Results
Free chitobiase activity versus body length was plotted for a11 species (D. magna, D.
pulex, Ceriodaphnia sp., D. galeatn, D. puticaria. D. dubia, Hgib berum, and marine copepods) .
Each of the species specific regressions (except for K. gibberum) were found to be significant,
p<0.000 1). Al1 species-specific regressions were also found to be significantly different fiom
each other (Figures 4.2-4.9, pc0.005). However, when the data, for al1 species (except H.
gibberurn, see Section 4.4) was pooled, a single significant relationship (logio [chitobiase
activity] = -1.19 +- 0.89 loglo [length], r2 = 0.79, p<0.0001) was found (Figure 4.10). In order to
test the validity of the pooled regression as a measure of any of the specific body Length-
chitobiase relationships, an F-test, pCO.05 was employed. Vaxiation about each individual
regression was compared to that of the overall regression. The F(12,307) = 15.07>2.3, therefore it
c m be concluded that one or more of the species specific regressions are better representative of
variation of each relationship than is the overall regression.
The linear increase of liberated MUF with tirne indicates substrate saturation for both
total (unfiltered) and crustacean (filtered) fractions of seawater (Figure 4.1 1). Cornparison of
filtered and unfiltered fractions suggested significant cell-bound chitobiase activity in the
ambient medium.
A difference in chitobiase activity was also observed between filtered and unfiltered
fieshwater samples, thus indicating significant cell-bound chitobiase activity. The activity of
chitobiase in sub-samples filtered at each time interval, diminished with t h e , while the activity
in the unfiltered fraction did not (Figure 4.12).
4.4 Discussion
The activity of chitobiase released by individual D a p h i a magna, D. pulex, and
Ceriodaphnia sp., followed a similar relationship with body length (Chapter 3). However, the
species-specific regressions were significantly different (Figures 4.1-4.3; p< 0.05). The status of
the additional cladoceran (D. galeata, D. dubia, D. pulicaria and H gibberurn) and copepod
species illustrate a similar condition. The slopes of individual regressions were found to differ
(Figures 4.4-4.9), but the regression through 6 freshwater cladoceran and 6 marine copepod
species examined was significant (Figure 4.10, p<O.001). It was also found that the overall
regression was not as effective in explaining variation of one or more species regressions than
was their own specific regression. Nevertheless, the overall regression was found to be both well
correlated and significant.
The utility o f the cornmon regression is significant in practical ternis. The most accurate
estimate of chitobiase activity liberated by a rnixed population would be obtained using each
species specific regression where each species is equally represented throughout the entire
population size range. An "ideal" population structure such as this, however, is rarely
representative of a natural community. As such, a simpler approach would be to apply the
cornmon relationship according to size, regardless of species composition. Furthemore, the size
ranges al1 of the species included in Figure 4.10 fa11 within the 95% confidence intervals
bounding the common regression. Thus, an estimate of chitobiase activity for a group of species
would only suffer significant error if the size range of a specific group extended beyond the
confidence limits of the common regression.
Investigation of chitobiase activity in other species that encompass the entire body size
range represented in this study, rnay M e r demonstrate the viability of this relationship, with
respect to the application presented in Chapter 3. The observed relationship between chitobiase
activity and body size for H gibberum was not significant and poorly correlated (r2 = 0.10,
Figure 4.8). Thus data for this species was not included in the pooled data set through which the
comrnon regression was applied. Chitobiase activity liberated by molting EL gibberum may be
more appropriateiy applied to another measure of body size such as weight. The exoskeletons the
other species studied are more calcified and IikeIy have a greater chitin content relative to H.
gibberum (personal communication N. Yan). The poor relationship to body length may be due to
the presence of the gel matrix, surrounding this cladoceran, which af5ords it some of the
structural integrity and protection of the more heavily calcified and ngid exoskeletons found in
daphnids and copepods.
The existence of a common relationship for 12 species of planktonic crustacea is
meaningful. Perhaps the most surprising aspect of this finding is that this relationship
encompasses both closely related and relatively disparate species. With respect to the
methodology presented in Chapter 3, a common relationship is important. It is the basis of an
assessment of development time, and hence growth rates in natural mixed populations of
crustacean zooplankton.
Given these initial resuits and the universality of chitobiase activity in crustaceans, it
appears that this relationship may hold for other species. Assuming a taxonomically conserved
fiee chitobiase relationship to body size, an in situ validation of this method depends on whether
the activity of chitobiase (and its rate of decay) in laboratory cultures, is detectable in the natural
environment.
Chitobiase attributed to molting crustaceans was present in samples removed from both
marine and freshwater environrnents. In both instances, activity in the total fraction (Le.
unfiltered) was higher than that in filtered fractions. In a rate of decay experiment, chitobiase
activity in the total fraction of freshwater sarnples remained constant while the activity in the
filtered sub-samples decayed with time (Figure 4.12). Therefore, it is important to distinguish
crustacean chitobiase from cell bound sources. The substrate, MUF-NAG, is not specific to only
the crustacean form of chitobiase. The potential implications of this lack of specificity demands a
discussion of the sources contributing to chitobiase activity in the total fraction (see Chapter 5).
Accordingly, it is important to recognize what the activity in fiitered and unfiltered sarnples
represent and the extent to which discrimination of chitobiase sources is needed.
References
Espie, P.J., and Roff, J-C. 1995. Characterization of chitobiase fiom Daphnia rnagrza and its
relation to chitin f lux Physiol. Zool., 68:727-748
McLaren, I.A., Sevigny, J-M., and Corkett, C.J. 1988. Body sizes, development rates and
Vrba, J., and Machacek, J. 1994. Release of dissolved extracellular P-N
acetylgIucosarninidase during crustacean molting. Limnol. Oceanogr., 3 9:7 12-7 16
Yan, N.D. and Macke, G.L. 1987. Improved estimation of the dry weight of Holopediurn
gibberurn (Crustacea, Cladocera) using clutch size, a body fat index and lake water total
phosphorus concentration. Cm. J. Fish. Aquat. Sci., 44: 3 8 1-3 89
O 1 O 20 30 40 50 60
Fluorescence (Perkin Elmer LS50 - fsu)
Figure 4.1. Cross calibration of fluorescence units (fsu) between Turner Designs (TD 7000) and Perkin Elmer Luminescence Spectrometer (LS 50). Fluorescence read on both machines at 360 nrn excitation and 450 nm emission. A dilution series of standard chitobiase in 700 pl of 0.2-pm filtered water incubated for 10 minutes with 0.4 mm01 methylumbelliferyl-N-acetyl-P-D- glucosaminide (MUF-NAG).
2.9 3 .O 3.1 3 -3 3 -3 3 -4 3 -5 3 -6
Log Body Length (pm)
Figure 4.2. Linear regression of chitobiase activity versus body length for Daphnia magna. Chitobiase activity is reported as nrnoles methylurnbelliferone (MUF) liberated in 10 minutes from 0.7 ml of medium. Individuals were incubated in 3.0 ml synthetic fkeshwater for 12 h and chitobiase activity assayed in aliquots removed from molted individuals. Where log [chitobiase activity] = -3.654 + 1.6445 log [body length]; r ' = 0.75, n = 95.
2.8 2.9 3.0 3- 1 3 -2 3 -3
Log Body Length (pm)
Figure 4.3. Linear regression of chitobiase activity versus body length for Daphnia pzdex. Chitobiase activity is reported as nrnoles methylurnbelliferone (MUF) liberated in 10 minutes fkom 0.7 ml of medium. hdividuals were incubated in 3.0 ml synthetic fieshwater for 12 h and chitobiase activity assayed in aiiquots removed fiom molted individuais. Where log [chitobiase activity] = -2.86 + 1 -433 log [body length]; r ' = 0.65, n = 52.
2.5 2.6 2.7 2.8 2.9 3 .O
Log Body Length (pm)
Figure 4.4. Linear regression of chitobiase activity versus body len-th for Ceriodaphnia sp. Chitobiase activity is reported as nrnoles methylurnbelliferone (MUF) liberated in 10 minutes fiom 0.7 ml of medium. Individuals were incubated in 3.0 ml synthetic fieshwater for 6 h and chitobiase activity assayed in aliquots removed from molted individuals. Where log [chitobiase activity] = -0.4748 + 06283 log Body length] ; r = O S 1 1, n = 44.
Log Body Length (pm)
Figure 4.5. Linear regression of chitobiase activity versus body length for Daphnia galeata. Chitobiase activity is reported as mo le s methylurnbelliferone (MUF) liberated in 10 minutes fiom 0.7 ml of medium. Individuals were incubated in 3.0 ml synthetic fieshwater for 6 h and chitobiase activity assayed in aliquots removed fiom molted individuals. Where log [chitobiase activity] = -2.26 + 1.29 log [body length]; r =0.60, n = 13.
Log Body Length (pm)
Figure 4.6. Linear regression of chitobiase activity versus body length for Daphnia pulicaria. Chitobiase activity is reported as nrnoles methylumbelliferone (MUF) liberated in 10 minutes fiom 0.7 ml of medium. Individuals were incubated in 3.0 ml synthetic fieshwater for 6 h and chitobiase activity assayed in aliquots removed fiom molted individuals. Where log [chitobiase activity] = -0.62 + 0.71 log pody length]; r =0.85, n = 9.
2.88 2.90 2.92 2.94 2.96 2.98 3.00 3 .O2
Log Body Length (pm)
Figure 4.7. Linear regression of chitobiase activity versus body length for Daphnia dubia . Chitobiase activity is reported as nmoles methylurnbelliferone ( N F ) liberated in 10 minutes fiom 0.7 ml of medium. Individuals were incubated in 3.0 ml synthetic Ereshwater for 6 h and chitobiase activity assayed in aliquots removed fkom molted individuals. Where log [chito biase activity] = -2.735 +1.39 log [body length]; r = 0.724, n= 7.
2.6 2.7 2.8 2.9 3 -0 3.1
Log Body Length (pm)
Figure 4.8. Linear regression of chitobiase activity versus length for Holopediurn gibbertirn . Chitobiase activity is reported as nmoles methylumbelliferone (MUF) Iiberated in 1 0 minutes from 0.7 ml of medium. Individuals were incubated in 3.0 ml synthetic freshwater for 6 h and chitobiase activity assayed in ahquots removed from molted individuals. Where log [chitobiase activity] = 0.69 +O. 167 log pody length]; r =O. 10 1 , n= 20.
Log Body Length (p)
Figures 4.9. Linear regression of chitobiase activity versus body length for marine copepods. Chitobiase activity is reported as mo le s methylurnbelliferone (MUF) liberated in 10 minutes fiom 0.7 ml of medium. lndividuals were incubated in 3.0 ml synthetic seawater for 6 to 9 h and chitobiase activity assayed in aliquots removed fiom molted individuals. Where log [chitobiase activity] =0.848 + 0.1589 log [body length]; r ' =0.458 . total n= 65 ((O), nauplii n= 20, (0) .
Figure 4.20. Linear regression of chitobiase activity versus body length for dl species pooled (Figures 4.2 -4.9, except 4.8). Chitobiase activity is reported as nrnoles methylumbelliferone (MUF) liberated in 1 O minutes fiom 0.7 ml of medium. Where log [chitobiase activity] = - 1.19 + 0.89 log [body length]; r =0.79, n= 285.
5 10 15 20 25 30 35 40 45
Time (minutes)
Figure 4.1 1. Tirne course for of the enzyme-substrate reaction of ambient chitobiase from seawater. Chitobiase activity expressed as nrnol of MUF liberated per unit time. Reaction was initiated with the addition of 0.4 mm01 MUF-NAG to 0-7 mi of native seawater collected fiom Passamaquoddy Bay, New Brunswick. Bars represent standard error of totd fiaction (n=3) and 0.2 pm filtered &actions (n=3). Linear regression of the total (unfiltered; a) activity versus time = 11.61 + 0.433 r2 =0.97. Linear regression of the crustacean (0 .2 -p filtered; O) activity versus tirne = 9.28 + 0.235 r2 = 0.92.
O 4 8 12 16 20 24
Tirne (hours)
Figure 4.12. Rate of decay of arnbient chitobiase in fieshwater collected fiom Plastic Lake, Dorset, Ontario. Aliquots (n=3) were passed through 60 p mesh and chitobiase activity assayed in 0.7 ml samples. Chitobiase activity is expressed as nmoles MUF liberated in 10 minutes. All reactions were initiated with the addition of 0.4 mm01 MUF-NAG. Samples (0.7 ml) removed fiom aliquots were either 0.2-pm filtered (O) or unfiltered (O) at each time interval. Bars represent standard errors of mean chitobiase activity.
Chapter 5. Framework for in situ applications, sampling protocols,
modifications, and conclusions
5.1 Applications. potential limitations. and modifications
Applications of the chitobiase assay proposed in Chapters 2 and 3 have been discussed in
terms of their feasibility as in situ measures of development tirne (Chapter 4). The following
sections of this chapter discuss the various natural sources of chitobiase in the water colurnn and
to what extent discrimination of these sources is needed. Further, a detailed discussion of molt
rate experiments and the potential for error due to handling stress are presented in order to
demonstrate some advantages and limitations of the method proposed in Chapter 2.
The rernaining sections are concerned with an in situ application of the method presented
in Chapter 3. Thus, an application of such a study requires discussion of preparatory
investigations, sampling protocols, potential biases, and specific modifications of the technique.
5.2. Free ambient chitobiase: What's in your sample?
Chitobiase activity may be of significant ecological importance in the water colwnn. A
number of studies (Le. Hoppe 1983; Vrba et al. 1992; Vrba et a1.1993) have demonstrated the
relative importance of chitin metabolism by a variety of unicellular organisms. There are at Ieast
three known sources of chitobiase resident in the water coIumn. These sources are chitobiase
bound to the ce11 membranes of certain bacterial species, that bound to various flagellates and
ciliates, and that released at ecdysis by crustaceans and other aquatic arthropods.
Cell-bound chitobiase and several other carbohydrate-hydrolyzing enzymes are employed
by some aquatic species of bacteria in DOC and FOC metabolism. Peptidoglycans of microbial
ce11 membranes are in part, composed of N-acetylglucosamine (NAG). Vrba et al. (1993)
demonstrated that the activity of chitobiase bound to the ce11 membranes of the flagellate, Bodo
saltans, and the ciliate, Cyclidium sp., was significantly correlated with total grazing rates on
bacteriai cultures of Aeromonas hydrophila and Alcaligenes xylosoxidans. Al1 three forms of the
enzyme were disthguished kinetically. The bacterial enzyme was of low substrate affinity (Km
>IO0 p o l MUF hour-' 1-'), while both the flagellate and ciliate chitobiase were of high substrate
-1 -1 affmity (Km < 1 pmoI MUF hour 1 ), the crustacean molting enzyme had intermediate Km
values, ranging from 45-65 p o l MUF hotir-' 1-' (Vrba et al. 1993; Vrba & Macacek 1994; Espie
& Roff 1995a ; Vrba et al. 1996; Oosterhuis et al. 2000).
Vrba, J.,. Simek, K., Pemthaler, J., and Psemer, R. 1996. Evaluation of extracellular, high
affînity B-N-acetylglucosaminidase measurements fiom Ereshwater lakes: An enzyme
assay to estimate protistan grazing on bacteria and picocyanobacteria. Micr. Ecol.,
32:8 1-99
Figure 5.1. Rate of arnbient chitobiase decay in 0.2 pn filtered and unfiltered water removed fiom Daphnia magna culture vesse1 in the laboratory. Aliquots were removed at tîme O and passed through a 60 pm mesh to remove animals. Aliquots were either filtered (O; n=3) or unfiltered (a; ~ 3 ) . Chitobiase activity was assayed in 0.7 ml sarnples every 3 to 6 hours from reactions with 0.4 rnrnol MUF-NAG for 1 O minutes. Bars represent standard errors of mean chitobiase activity.
500 1 O00 1500 2000 2500 3000 3500 4000
Body LengSi (pm)
Figure 5.2. Chitobiase activity in non-apolytic homogenates of Duphnia magna (n=16). Activity is expressed as nmoles methylurnbelliferyl (MUF) liberated in 10 minutes. Animals were homogenized in 3.0 ml of citrate-phosphate buffer, pH 5.5. Fluorescence read at 360 nm excitation and 450 nm emission on Perkin Elmer (LS 50) Spectrorneter: regression of activity versus body length (pm); Activity = -1 39.33 + 0.754 (body length), 8 = 0.75, p<0.000 1.
500 1 O00 1500 2000 2500 3 O00 3500 4000.
Body Length (pm)
Figure 5.3. A cornparison of chitobiase activity (nmoles methylurnbelliferyl (MUF) liberated in 10 min) liberated into the medium (O) and resident in the apolytic space ( a ) of homogenates of Daphnia magna.
Appendices.
Appendix 1 Synthetic fieshwater (as per Roff et al. 1994). AU reagents diluted in Nanopure filtered water. Bracketed values indicate composition of synthetic freshwater used for incubations of Daphnia magna.
Appendix 2. Effect of length of reaction time on hydrolysis of methylumbelliferyl-N-acetyl-PD- glucosaminide (MUF-NAG) with 0.7 ml of medium fkom individual Daphnia magna (2,000- 2,100 pm) incubated for 6 h (n = 7 moIted (O), versus n = 7 non-molted (O)). Linear regressions of chitobiase activity (nrnol MUF liberated) versus tirne are:
= Molted, a = 4.013, b = 24.964, r2 = 0.8142, p < 0.0001. O =Non-molted, a = 0.279, b= 13.831, r2 = 0.335, n.s. Bars represent standard errors of mean chitobiase activity of molted and non molted individuals.
O 1 O 20 30 40 50 60 70
Tirne (minutes)
Appendix 3a. Fluorescence versus time for different substrate concentrations of methylumbelliferyl-N-acteylglucosamine (MIE-NAG). Curves are fitted with straight lines. Substrate (3 1.124 nmol) was incubated with 2 pl of standard chitobiase (Sigma Chemical Co.) in 150 pl citrate-phosphate buffer (CPB, 0.15 M, pH 5.5) until maximum fluorescence was observed. Change in fluorescence represents hydrolysis of MUF-NAG to rnethylurnbelliferone (MUF) and N-acetylglucosarnine (NAG) .
O 20 40 60 8 0 1 O0 120
Tirne (minutes)
Appendix 3 b. Fluorescence versus time for different substrate concentrations of methy lumbelli feryl-N-acte y lg lucosamine (MW-N AG) . Curves are fitted with straight lines. Substrate (48 m o l ) was incubated with 2 pl of standard chitobiase (Sigma Chemical Co.) in 150 pl citrate-phosphate buffer (CPB, 0.15 M, pH 5.5) until maximum fluorescence was observed. Change in fluorescence represents hydrolysis of MüF-NAG to methylumbelliferone (MUF) and N-acetylglucosamine (NAG).
O 20 40 60 8 O 1 O0 120 160
Time (minutes)
Appendix 3c. Fluorescence versus time for different substrate concentrations of methylurnbelliferyl-N-acteylglucosamine (MUF-NAG). Curves are fitted with straight lines. Substrate (75 nrnol) was incubated with 2 pl of standard chitobiase (Sigma Chernical Co.) in 150 pl citrate-phosphate buffer (CPB, 0.15 M, pH 5.5) until maximum fluorescence was observed. Change in fluorescence represents hydrolysis of MUF-NAG to methylumbelliferone ( N F ) and N-acetylglucosamine (N AG) .
O 2 0 4 0 60 8 0 100 120 140 160
Time (minut es)
Appendix 3d. Fluorescence versus time for different substrate concentrations of rnethylumbelliferyl-N-acteylglucosamine (MUF-NAG). Curves are fitted with straight lines. Substrate (1 00 m o l ) was incubated with 2 pl of standard chitobiase (Sigma Chernical Co.) in 150 pl citrate-phosphate buffer (CPB, 0.15 M, pH 5.5) until maximum fluorescence was observed. Change in fluorescence represents hydrolysis of MUF-NAG to methylurnbelliferone (MüF) and N-acetylglucosamine (NAG).
O 20 40 60 8 O IO0 120 140 160 180 200
Tirne (minutes)
Appendix 3e. Fluorescence versus time for different substrate concentrations of methylurnbelliferyl-N-acteylglucosamine (MUF-NAG). Curves are fitted with straight lines. Substrate (175 nmol) was incubated with 2 pl of standard chitobiase (Sigma Chernical Co.) in 150 pl citrate-phosphate buffer (CPB, 0.15 M, pH 5.5) until maximum fluorescence was observed. Change in fluorescence represents hydrolysis of MUF-NAG to methylumbelliferone (MUF) and N-acetylglucosamine (NAG).
O 50 1 O0 150 200 250
Tirne (minutes)
Appendix 3f. Fluorescence versus time for different substrate concentrations of methylumbellifery 1-N-acteylglucosamine (MUF-NAG) . Curves are fitted with straight lines. Substrate (250 nrnol) was incubated with 2 pl of standard chitobiase (Sigma Chernical Co.) in 150 pl citrate-phosphate b a e r (CPB, 0.15 M, pH 5.5) until maximum fluorescence was observed. Change in fluorescence represents hydrolysis of MUF-NAG to methylurnbelliferone (MUF) and N-acetylglucosamine (NAG).
O 5 O 1 O0 150 200 250 300
Methyhnbelliferone produced (moles)
Appendix 3g. Linear regression of fluorescence versus methylumbelliferone produced (fluorescence values detemined as maximal fluorescence fiom Appendices 3a to f)- FIuorescence values are expressed as chitobiase activity (nmol MUF liberated per unit time) as per the regression parameters: Fluorescence = -39.8 + 3.18 ( m o l e s MUF produced), r2 = 0.97.
Time (hours)
Appendix 4. Time course for decay of chitobiase (unfiltered) released into medium by individuai Daphnia magna (n=5). Bars represent standard error of mean percent chitobiase activity versus tirne. Percent chitobiase activity expressed relative to Time=O (100% activity).
Time (hours)
Appendix 5. T h e course for decay of chitobiase released into medium by individual Daphnia magna (n=6) into water that has been 0.2-pm fdtered. Bars represent standard error of mean percent chitobiase activity versus time. Percent chitobiase activity expressed relative to Time=O (1 00% activity).
O 2 4 6 8 10 12 14
T h e (days)
Appendix 6. Mean percent chitobiase activity remaining i n 0.2-prn filtered samples of incubation medium exposed to molted Daphnia magna versus time (days). Percent chitobiase activity expressed relative to Time=O (100% activity). Bars represent mean standard error (n = 12).
Body Iength (pm)
Appendix 8. Mean size fiequency distribution of subsamples (n=3) fiom Daphnia rnugna laboratory culture. Total culture volume = 10.013 L. Total subsample volume = 268 ml. Total estimated population = 1 868 individuals.
Body length (pm)
Appendix 9. Mean size fiequency distribution of subsamples (n=3), in the mixed laboratory culture of Daphnia magna - Ceriodaphnia sp. Total culture vesse1 volume = 6.934 L. Subsample volume = 268 ml. Total estimated population = 4502 individuals.
O 500 1 O00 1500 2000 2500 3000 3 500
Body Length (pm)
Appendix 10. Intermolt period versus body length for three cladoceran species. Development times @ours) for Ceriodaphnia sp. (O), Daphniaplex ( A ) , and Daphnia magna (O). (n = 20-25 for each size class). D = 26.45 + 0.02 1 (body length) ; 8 = 0.9 1, p < 0.000 1.