METABOLISM OF AQUATIC INVERTEBRATES
Metabolism of aquatic invertebrates
Introduction
Measurement of the oxygen consumed by an organism can be used as direct proxy for estimating its metabolic rate.
Where:
M = O x 20.11
where M = the metabolic rate (J h–1) and O = O2 consumed (cm3 h–1).
In this practical, you will use the Winkler method to determine the respiration rate and hence metabolic rate of three different aquatic invertebrates, the Mystery Snail (Pomacea diffusa), the Common Yabby (Cherax destructor) and Freshwater Mussels (Velesunio ambiguus). For the Mystery Snail you will also measure and calculate their Q10.
The Winkler determination of dissolved oxygen (DO), utilises the fact that manganous hydroxide will react with dissolved oxygen to produce a tetravalent manganese compound. In the presence of acid potassium iodide an equivalent quantity of iodine is liberated (equivalent to the DO content), which can be titrated against a standard solution of sodium thiosulphate. With a starch indicator, the end point is a clear solution.
Mn2+ + 2OH– Mn(OH)2
2Mn(OH)2 + O2 2MnO(OH)2
MnO(OH)2 + 4H+ + 3I– Mn2+ + I3– + 3H2O
I3- + 2S2O3 2- 3I- + S4O62-
Metabolism of aquatic invertebrates
Methods
Preparation of animals
Select 3 snails, 1 yabby and 1 mussel from the buckets on the front bench and place them into different (yabby vs. molluscs) containers of water. For each specimen, measure their shell/carapace width, length and height, weigh them (wet weight) after blotting off excess water and return them to the container filled with water.
Preparation of respiration chambers (pickling jars)
Take six pickling jars and lids from the front.* Smear the rim of each lid with a small amount of Vaseline to ensure an airtight seal. Using a measuring cylinder, accurately fill three jars with 250 ml of seawater from the aerated water bucket/water bath at 20 oC, one jar with 250 ml of water at 25 oC and one jar with water at 30 oC. You must be accurate. Once the jars are filled move them carefully with your animals (in their bucket) to the Nitrogen cylinder. Place a yabby, a mussel and a snail separately, in one of the jars at 20 oC (n = 3), one snail in the jar filled with water at 25 oC and one snail in the jar filled with water at 30oC.
Once the animals are in their jar, flush the head space with nitrogen, screw the cap on and record the time the jar was capped. Repeat this for the 4 remaining jars, such that you end up with 6 capped jars, one containing nothing (control) and each of the others containing an individual animal. Leave the animals to respire for 60+ minutes (so long as we record the time the animals were capped for, it doesn’t matter if it is 60 minutes or 80 minutes etc.).
*NB depending on the size of the animals, we may have to use larger containers and hence increase the volume of water used.
Metabolism of aquatic invertebrates
Determination of dissolved oxygen (titration)
You must wear gloves and goggles/glasses.
For your first few attempts, practice using water taken from the aerated water bucket.
- Using the 60 ml syringe, fill the syringe including the dead space with 1 ml of manganous sulphate.
- Carefully, without contamination and/or drawing in air suck in 1 ml of alkaline iodide solution.
NB the exact amounts are not that important, so long as you have at least 0.5 ml of each and know the exact volume of both components.
- With your finger over the tip of the syringe place the tip in the jar of water to be measured and suck in a further 50 ml of water. NB The total volume is not as important as knowing exactly how much you have sucked it.
- Once you have pulled in sufficient water, place your finger over the tip of the syringe and carefully mix the content by repeated inversion (turning upside down and back again).
- Expel the total contents of the syringe (including the precipitate that has developed) into an Erlenmeyer flask.
- Using the automatic dispenser, add 1 ml of concentrated sulphuric acid and very carefully mix the contents (swirl). The precipitate will dissolve.
Concentrated sulphuric acid will immediately burn you if you manage to spill it on yourself, so be very careful and take care.
- Titrate the contents of the flask with the sodium thiosulphate sulphate solution until you obtain a pale straw (weak urine) colour, add a drop (0.5 ml) of starch solution and continue to titrate until colourless.
- End products can be disposed of down the sink followed by rinsing with water.
You will follow exactly the same procedure for your experimental jars. Place the animals into the rescue bucket, once you have drawn your water.
Metabolism of aquatic invertebrates
Calculation of dissolved oxygen
ml O2 l-1 = n x F x 1000 x 22,400
f x 4 x 1000
Where n = ml of thiosulphate used in the titration, F = the normality of the thiosulphate (ask) and f = the volume of water taken, i.e. drawn into the syringe.
If you have calculated the DO of your control, you can now use this to calculate the amount of oxygen consumed by the animals (control DO – experimental DO) per unit time (I would use per 24 h). For comparison with the literature and between species, i.e. express this as oxygen consumption per g (wet weight) of the whole animal per 24h
Metabolism of aquatic invertebrates
Metabolism of aquatic invertebrates
Results
1x plot for respiration v morphological measurement @ 20C for all three species, with equations.
1x plot for respiration across three temps for snails OR yabbies, with Q5’s Calculated standardised respiration rates per gram for all three species.
Are given in excel sheet PLEASE SEE THE attached DATA
Requirements for practical write-up (results/discussion)raw data
- For the class data plot one of the morphological measurements (height, length, width, weight) against oxygen consumed by the species maintained at 20 oC, and plot by eye a line of best fit for each species (you could put all species on the same plot). Snails, yabbies and mussels
- Calculate the slopes of the lines (gradients) and use it to compare between the species. Are there differences and if so why?
- For the snails, using the class data, plot their respiration (oxygen consumed) against incubation temperature and calculate a Q10 for the species. Snails OR yabbies
- Using the class data calculate a mean respiratory rate per gram of whole animal (wet weight) and convert this to metabolic rate. Is there a difference between the species? How do your metabolic/respiratory rates compare with those in the literature? Standarised respiration
- How could you improve the experiment? What do you think are the biggest sources of error?
Example Introduction
Start broad relevant to your aim, outline importance and narrow to your aim, finishing your introduction with your aim. Pedal mucus is energetically expensive to produce, accounting for 9 – 74% of the energy assimilated by gastropods (see Davies and Hawkins 1998 for review) and hence the advantage to a gastropod in utilising pedal mucus for an additional function other than locomotion is evident. For example, the pedal mucus produced by Littorina littorea (Linnaeus, 1758) during locomotion and left behind on a surface can act as provendering agent with its subsequent ingestion once it has entrapped particulate matter (see Imrie 1992).
The phenomenon of shell wiping, where a layer of pedal mucus is deposited on the surface of the shell by the foot, has been recorded in the calliostomatid gastropods Calliostoma zizyphinum (Linnaeus, 1758), C. granulatum (Born, 1778), C. ligatum(Gould, 1849) and C. canaliculatum (Lightfoot, 1786) (see Theisen 1972 for details of a bivalve equivalent). For the first two species it is believed to act as a supplementary feeding strategy through provendering and its subsequent ingestion, accounting for 20% of the daily energetic requirement of C. zizyphinum (Holmes et al. 2001), and for all species as a mechanism to reduce shell fouling (Keen, 1975; Jones 1984; Holmes et al. 2001; Holmes and Duncan 2007).
In addition, for C. canaliculatum shell wiping is also thought to act as a mechanism to reduce predation, where the foot smears the surface of the shell both with pedal mucus and an exudate from the hypobranchial gland that is noxious to predatory ophiuroids (Bryan et al. 1997). Furthermore, it is worth noting that although shell wiping has not been observed in C. gloriosum (Dall, 1871), animals can be found with a layer of mucus, sufficient to blur surface features, covering their shell (Keen 1975).
Given that: 1) there are no published records for shell wiping occurring in any other gastropod species, Calliostomatidae or otherwise; 2) the direct application of pedal mucus to a surface serving a function other than locomotion, has not been noted for any other gastropods; and 3) shell wiping can serve different functions within the same genus, it is possible that shell wiping is a feature unique to, and solely exhibited by, members of the Callisotoma genus. The aim of this paper is to detail shell wiping behaviour serendipitously observed in a fifth calliostomatid species, Calliostoma (Otukaia) alertae B. Marshall, 1995.
Example Materials and Methods
The concentration of iron in the haemolymph of six invertebrates (a starfish, a xxx) was measured by taking 10 ml of haemolymph from each organism and measuring the iron content using
