and maintained in the laboratory if they are provided with adequate food and if the water is kept cool, pure, and well oxygenated. The latter two conditions are ...
CHAPTER 40
MAINTENANCE OF SHREDDERS IN THE LABORATORY FERNANDO COBO Departmento de Biología Animal, Facultad de Biología, Universidad de Santiago de Compostela, 15782, Santiago de Compostela, Spain.
1. INTRODUCTION Shredders are aquatic invertebrates whose mouth parts are adapted for feeding on large particles of organic matter such as decomposing leaves. Most shredders from temperate areas are insects (primarily Plecoptera, Tipulidae, Limnephilidae and other Trichoptera) and crustaceans (Amphipoda, Isopoda). Shredders can be grown and maintained in the laboratory if they are provided with adequate food and if the water is kept cool, pure, and well oxygenated. The latter two conditions are easy to achieve in an aquarium with a good filter and adequate aeration. Low temperature is more difficult to attain if expensive equipment such as temperature-controlled rooms or chambers, large cooled incubators orr low-temperature aquaria are unavailable. Some species also need water flow to ensure high survivorship, which poses an additional constraint in the design of a maintenance system. Studying invertebrates in the laboratory provides biologists with an opportunity to work in a controlled environment. A variety of techniques have been described (Allegret & Denis 1972, Armitage & Davis 1989, MacKay 1981, Craig 1993). They range from simple Petri dishes to recirculating streams with complex pumps and cooling systems. Several systems have been constructed mainly for taxonomic or behavioural studies (e.g. Wiggins 1959, Resh 1972, Wiley & Kholer 1980, Smith 1984, Keiper & Foote 1996). If reproduction is successful, large numbers of individuals can be reared and then used in experiments aimed at assessing the involvement of shredders in decomposition (e.g., determination of consumption rates, food preferences, gut enzyme activities). Moreover, some stream invertebrates can only be accurately identified as adults or from exuviae. Consequently, larval stages often have to be reared if identification to the species level is sought (Philipson 1953, Hiley 1969, Bjarnov & Thorup 1970). 291 M.A.S. Graça, F. Bärlocher & M.O. Gessner (eds.), Methods to Study Litter Decomposition: A Practical Guide, 291– 296. © 2005 Springer. Printed in The Netherlands.
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This chapter describes an inexpensive versatile laboratory system for maintaining and rearing stream macroinvertebrates. The system has been used successfully to grow Trichoptera, Plecoptera, Ephemeroptera and Chironomidae from eggs to adults. The reared specimens off these taxa have been collected in cold, fast flowing waters. 2. DESIGN AND MATERIALS A refrigerator for low-temperature storage of meals cooked in advance as commonly used in catering is used to keep the invertebrates in a temperature-controlled environment (Fig. 40.1). The refrigerator should have a stainless-steel tray that can be used as an aquarium. The tray should be located on the top of the refrigeration circuit that is controlled by a thermostat (Fig. 40.1 A) and preferably be covered by a plexiglass case. Two movable front panels allow manipulation of the invertebrates. The plexiglass enclosure creates air space for emerged adults. At the top of the plexiglass enclosure, two fluorescent tubes of the “day-light” type (8 W) are installed (Fig. 40.1 B). The electrical circuits for the thermostat and fluorescent tubes should be kept separate so that the tubes can be connected to a programming device controlling the light-dark cycle. Water leaves the stainless-steel tray by a plastic tube passing through a hole drilled in the tray and the case. A second ttube enters the refrigerator through another hole drilled in the plexiglass cover. The tube is blocked at its end, perforated along the section allocated inside the refrigerator and fixed above the water level so that incoming water cascades over the tray, creating turbulence and thus ensuring oxygenation (Fig. 40.2). An aquarium pump provided with a filtering system circulates and purifies the water (Fig. 40.1 C).
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Figure 40.1. Schematic representation of the closed culture system. A: Refrigerator; B: Tube sealed at the end and with small holes inside the chamber; C: Fluorescent tubes. D: Hole in the tray for water exit; E and F: outflow and inflow tubes for water circulation and water pump with filtering system; G: Power connections (light and refrigerator); H: Individual enclosure.
Additionally, small enclosures (Fig. 40.1 H) can be used to rear invertebrates individually within the refrigerator. Enclosures can be constructed using a strong coarse plastic mesh as a framework and a fine plastic mesh lining the inner sides. The size of the inner mesh can range from 250—500 µm, depending on size of the individuals. The top of the enclosures can be covered with plastic lids or Petri dishes, in case individuals are to be collected individually from each chamber. The enclosures are placed directly over the steel tray. It is often important to keep individual invertebrates separate to avoid predation; this allows maintaining several species simultaneously. Moreover, some pupae and final instar larvae can use the plastic mesh of enclosures to climb out of the water and emerge. The plastic lids or Petri dishes prevent adults from leaving the enclosures. When rearing Trichoptera, materials suitable for the construction of the larval case have to be added (e.g. sand, small pieces of wood, moss). The water used to rear invertebrates should ideally be filtered stream water. However, when this is not possible, artificial stream water can be used. Many formulas have been proposed (e.g. Graça et al. 1993). The following composition has ensured high survival rates for many species in our laboratory: 35 mg NaCl, 2 mg KH2PO4, 61.5 mg MgSO4, 36 mg CaCl2, 5 mg NaHCO3 and 1.6 mg FeCl3 per litre.
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Routine maintenance of the system consists of adding distilled water (UV irradiated or autoclaved; pH adjusted to values observed in streams). The stainlesssteel tray and the filter must be cleaned, and the water must be renewed about every two months.
Figure 40.2. Enclosures (arrows) s with invertebrates in the tray of the culture system
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3. REFERENCES Allegret, P. & Denis, C. (1972). Dispositif pour l’élevage d’insectes aquatiques a température constante. Annales d’Hydrobiologie, 3, 65-67. Armitage, P. & Davies, A. (1989). A versatile laboratory stream with examples of its use in the investigation of invertebrate behaviour. Hydrobiological Bulletin, 23, 151-160. Bjarnov, N. & Thorup, J. (1970). A simple method for rearing running-water insects, with some preliminary results. Archiv für Hydrobiologie, 67, 201-209. Craig, D.A. (1993). Hydrodynamic considerations in artificial stream research. In G.A. Lamberti, & A.D. Steinman (eds.), Research in Artificial Streams: Applications, Uses and Abuses (pp. 324-327). Journal of the North American Benthological Society, 12, 313-384. Graça, M.A.S., Maltby, L. & Calow, P. (1993). Importance of fungi in the diet of Gammarus pulex (L.) and Asellus aquaticus (L.): II. Effects on growth, reproduction and physiology. Oecologia, 96, 304309. Hiley, P.D. (1969). A method of rearing Trichoptera larvae for taxonomic purposes. Entomologist's Monthly Magazine, 105, 278-279. Keiper, J.B. & Foote, B. A. (1996). A simple rearing chamber for lotic insect larvae. Hydrobiologia, 339, 137-139. MacKay, R.J. (1981). A miniature laboratory stream powered by air bubbles. Hydrobiologia, 83, 383385. Philipson, G.N. (1953). A method of rearing trichopterous larvae collected from swift-flowing waters. Proceedings of the Royal Entomological Society of London, Series A, 28, 15-16. Resh, V.H. (1972). A technique for rearing caddisflies (Trichoptera). Canadian Entomologist, 104, 19591961. Smith, M.H. (1984). Laboratory rearing of stream-dwelling insects. Antenna, 8, 67-69. Wiggins, G.B. (1959). A method of rearing caddisflies (Trichoptera). Canadian Entomologist, 91, 402405. Wiley, M.J., & Kholer, S.L. (1980). Positioning changes of mayfly nymphs due to behavioural regulation of oxygen consumption. Canadian Journal of Zoology, 58, 618-622.
