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Cal Fremling Papers Cal Fremling Archive

1967

Methods for mass-rearing Hexagenia mayflies (Ephemeroptera: Ephemeridae)

Cal R. Fremling Winona State University

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Recommended Citation Fremling, Cal R., "Methods for mass-rearing Hexagenia mayflies (Ephemeroptera: Ephemeridae)" (1967). Cal Fremling Papers. 20. https://openriver.winona.edu/calfremlingpapers/20

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Made in United States of America Reprinted from TRANSACTIONS OF THE AMERICAN FISHERIES SOCIETY Vol. 96, No. 4, 2 October 1967 pp. 407-410

Methods for Mass-Rearing Hexagenia Mayflies ( Ephemeroptera: Ephemeridae)1

INTRODUCTION Hexagenia bilineata and Hexagenia limbata mayfly nymphs are important food organisms for Mississippi River fishes (Hoopes, 1960) and they seem to be excellent indicators of general water quality (Fremling, 1964a). The general life histories of Hexagenia mayflies are well known (Needham, Traver, and Hsu, 1935; Hunt, 1953; Fremling, 1960). The burrowing nymphs construct U-shaped respira­ tory tubes in the muddy bottoms of lakes and rivers where they ingest mud, organic detritus, algae and bacteria. Hexagenia nymphs re­ quire from three months to a year to mature in the Upper Mississippi River, whereupon they rise to the surface, usually at night, cast their nymphal exuviae and emerge as sub- imagoes. Subimagoes rest in the shade along the river bank until the following afternoon when a final molt occurs and the imagoes emerge. Mating occurs aerially along the shoreline at dusk and the females return to the river where each alights on the surface and deposits two egg packets, each of which contains about 4,000 eggs. The eggs sift downward to the river bottom where most of them hatch in 10-12 days, if conditions are favorable. Male and female imagoes die within hours after they have mated. Hexagenia mayflies tend to emerge en masse, and river residents are accustomed to nuisance

1 Supported in part by National Science Founda­ tion Grant G-17498 and by Water Pollution Control Administration Grant WP-009871-01. 408 SHORT PAPERS AND NOTES

problems caused by the insects during periods are weighted down by an accumulated mass of maximum emergence. Analyses of over of other mayflies. 500 mayfly collections along the Upper Mis­ Large numbers of eggs are most easily sissippi River indicate that mass emergences collected by parking an automobile along the of H. bilineata mayflies tend to occur at in­ water's edge and by placing a water-filled tub tervals of about 6 to 11 days from mid-June beneath the headlights. Insects which are at­ through mid-August (Fremling, 1964b). tracted to the headlights fall into the tub We have studied the rhythmic emergence where they lay their eggs. When the layer phenomenon for the past 8 years to determine of insects becomes too thick, the insects are the environmental factors which influence it. skimmed off to make room for more. On Much of our investigation has been done in­ one occasion we parked our automobile on a doors under controlled conditions. The pur­ little-used bridge at Winona, Minnesota, and pose of this paper is to present the techniques collected 5.5 liters of eggs (ca. 345 million) developed to maintain large laboratory pop­ in one hour and 40 minutes. ulations of Hexagenia mayflies. Eggs may also be extracted from adults reared in the laboratory. It is unlikely, how­ COLLECTION OF EGGS ever, that mating will occur under laboratory Female imago mayflies are most easily col­ conditions because H. bilineata mayflies have lected from beneath lights along the water's an elaborate mating behavior pattern which edge. Mature females are usually more com­ involves swarming and sight recognition of mon around lights than are imago males or the female by the male (Fremling, 1960). subimagoes of either sex. Virtually all of the Unmated female imagoes have been observed mayflies collected beneath bridge lights are to lay eggs in laboratory aquaria, but unsuc­ inseminated imago females. Apparently these cessful attempts have been made on 12 oc­ have been diverted to the light as they flew casions to hatch H. bilineata eggs partheno- upriver to lay their eggs. genically. Fertile eggs may be obtained If small amounts of eggs are desired, it is readily, however, by artificial insemination. usually simplest to return the gravid females Eggs stripped from female imagoes or subi­ to the laboratory. They may be transported magoes are mixed with the macerated sexual easily by picking them up by the wing tips elements of the male in the insect's own body and placing them in a large inflated paper fluids, as described by Needham (1935). If bag. If egg collection must be postponed for insects arise from the rearing tank in very some reason, the bag and its contained insects small numbers, and if males are not im­ should be refrigerated to slow the metabolism mediately available, females may be kept for of the insects and thus lengthen their life up to two days in paper bags in the refrigerator span. at 10 C. Females are dropped, a few at a time, into a large water-filled funnel, the stem of which STORAGE AND INCUBATION OF EGGS is fitted with a rubber tube and a pinch Eggs are conveniently stored and incubated clamp. As they struggle on the surface of in water-filled polyethylene bags which mea­ the water, the females release their eggs which sure 10 X 16 X .0015 inches. The eggs are sink slowly into the stem of the funnel where able to respire because polyethylene is perme­ they may be tapped off in the desired able to oxygen and carbon dioxide. Up to 3 cc quantities. of eggs may be stored per bag for 120 days Imago females release their eggs most at 12 C with maximum viability (Flattum, readily when they are lying right side up on 1963). After 120 days, the eggs will begin to the water surface with their outstretched wings hatch at this temperature if oxygen is available adhering to the surface film. They have dif­ to them. ficulty ovipositing unless their wings are im­ We have stored H. bilineata eggs for 380 mobilized in some manner. Thus, they also days at 12 C by immersing the bags in water lay eggs when they are caught in spider webs, which contained soured milk. Because milk or if they are stuck to wet pavement or if they has a high bio-chemical-oxygen demand, the SHORT PAPERS AND NOTES 409 dissolved oxygen concentration of the ambient strong rubber bands. The bags are then hung water varied from 0.1 to 0.3 ppm during the in slots which are cut in a shelf. A cork 380 days. The viability of the eggs decreased stopper is tacked to the center part of each steadily with time. The normal time required Kerr cover which functions as a cover for for the eggs to hatch after removal from this the bag. cold anaerobic environment was increased by Nymphs ingest quantities of mud, detritus, 4 days at 22 C. algae, and bacteria principally from the mud Four liters of eggs in a small amount of surface, using their fore-legs to carry the water have been stored anaerobically at 12 C material to their mouths. As the nymphs for 79 days in a sealed jar. The water turned excavate the mud for food, their nymphal black and smelled strongly of hydrogen sulfide. burrows constantly change positions. Nymphal Viability decreased steadily with time and feeding activities and respiratory movements only 5% of the eggs were viable after 79 cause the water to become turbid. Indeed, days. No eggs remained viable longer than highly turbid water indicates a vigorous pop­ 110 days. ulation. Eggs may be easily incubated in the poly­ A constant algae bloom is maintained by ethylene bags in which they were stored. A hanging 250-watt incandescent lights, with black counter top is an ideal place for in­ reflectors, within one foot of the water sur­ cubation because the newly-hatched nymphs face. The lights also maintain the tanks at are white and their movements may be dis­ temperatures which exceed room temperature. cerned with the naked eye. The constant burrowing activities of the The rate of development of Hexagenia eggs nymphs make the soil nutrients available for is dependent upon temperature. A cold period algae growth and the algal rain provides a is not necessary as it is for some other may­ constant source of food. flies such as Ephoron album (Britt, 1962). Large, illuminated reservoir tanks are filled H. bilineata eggs hatch in 12 days at 22 C with tap water to which inorganic fertilizer and in 8 days at 32 C. If the eggs are clumped, is added (20 gm of Armour Vertegreen 10- hatching time is greatly prolonged. The eggs 10-10 per 50 gallons of water) to promote an on the outside of the clumps hatch first be­ algae bloom. This algae-rich water is used cause they are best oxygenated. to replace water lost through evaporation from the rearing tanks. MAINTENANCE OF NYMPHAL POPULATIONS Eggs may be placed directly into the rearing Nymphs have been successfully reared in chamber for incubation or they may be galvanized stock-watering tanks, galvanized hatched and then added. In the latter case wash tubs, various glass aquaria, and in large it is advisable to place the unopened bags full polyethylene bags. The containers are filled of newly-hatched nymphs in the rearing tank to a depth of 15 cm with rich garden soil for an hour before opening to equalize the which is prepared by spading in moderate water temperatures. amounts of thoroughly composted hay. In­ Nymphal growth is negligible at 14 C, but organic fertilizer (Armour Vertegreen 10-10- the rate increases with temperature. Only 79 10) is mixed in at the rate of 100 gm per m2 days were required to develop newly-hatched of soil area. We have successfully used the H. bilineata nymphs to adults in a tank which same soil for five years by enriching it in was maintained between 24 and 27 C. the preceeding manner. When nymphs are crowded, they exhibit Enough water is added to fill the con­ differential growth rates. The larger nymphs tainers. Constant aeration is provided to main­ apparently inhibit the growth of smaller tain a high level of dissolved oxygen in the nymphs. In one instance a crowded population water. Polyethylene bags are well suited for which was initiated at about 7000 nymphs isolated individuals because the bags are in 2.3 m2 of substrate finally produced 479 permeable to oxygen and thus require no adult insects. The adults matured at varying bubbler. We attach Kerr jar covers upside rates, however, the last adults emerging 327 down in the mouths of polyethylene bags with days after the first ones. Differential growth 408 SHORT PAPERS AND NOTES SHORT PAPERS AND NOTES 409 problems caused by the insects during periods are weighted down by an accumulated mass dissolved oxygen concentration of the ambient strong rubber bands. The bags are then hung of maximum emergence. Analyses of over of other mayflies. water varied from 0.1 to 0.3 ppm during the in slots which are cut in a shelf. A cork 500 mayfly collections along the Upper Mis­ Large numbers of eggs are most easily 380 days. The viability of the eggs decreased stopper is tacked to the center part of each sissippi River indicate that mass emergences collected by parking an automobile along the steadily with time. The normal time required Kerr cover which functions as a cover for of H. bilineata mayflies tend to occur at in­ water's edge and by placing a water-filled tub for the eggs to hatch after removal from this the bag. tervals of about 6 to 11 days from mid-June beneath the headlights. Insects which are at­ cold anaerobic environment was increased by Nymphs ingest quantities of mud, detritus, through mid-August (Fremling, 1964b). tracted to the headlights fall into the tub 4 days at 22 C. algae, and bacteria principally from the mud We have studied the rhythmic emergence where they lay their eggs. When the layer Four liters of eggs in a small amount of surface, using their fore-legs to carry the phenomenon for the past 8 years to determine of insects becomes too thick, the insects are water have been stored anaerobically at 12 C material to their mouths. As the nymphs the environmental factors which influence it. skimmed off to make room for more. On for 79 days in a sealed jar. The water turned excavate the mud for food, their nymphal Much of our investigation has been done in­ one occasion we parked our automobile on a black and smelled strongly of hydrogen sulfide. burrows constantly change positions. Nymphal doors under controlled conditions. The pur­ little-used bridge at Winona, Minnesota, and Viability decreased steadily with time and feeding activities and respiratory movements pose of this paper is to present the techniques collected 5.5 liters of eggs (ca. 345 million) only 5% of the eggs were viable after 79 cause the water to become turbid. Indeed, developed to maintain large laboratory pop­ in one hour and 40 minutes. days. No eggs remained viable longer than highly turbid water indicates a vigorous pop­ ulations of Hexagenia mayflies. Eggs may also be extracted from adults 110 days. ulation. reared in the laboratory. It is unlikely, how­ Eggs may be easily incubated in the poly­ A constant algae bloom is maintained by COLLECTION OF EGGS ever, that mating will occur under laboratory ethylene bags in which they were stored. A hanging 250-watt incandescent lights, with Female imago mayflies are most easily col­ conditions because H. bilineata mayflies have black counter top is an ideal place for in­ reflectors, within one foot of the water sur­ lected from beneath lights along the water's an elaborate mating behavior pattern which cubation because the newly-hatched nymphs face. The lights also maintain the tanks at edge. Mature females are usually more com­ involves swarming and sight recognition of are white and their movements may be dis­ temperatures which exceed room temperature. mon around lights than are imago males or the female by the male (Fremling, 1960). cerned with the naked eye. The constant burrowing activities of the subimagoes of either sex. Virtually all of the Unmated female imagoes have been observed The rate of development of Hexagenia eggs nymphs make the soil nutrients available for mayflies collected beneath bridge lights are to lay eggs in laboratory aquaria, but unsuc­ is dependent upon temperature. A cold period algae growth and the algal rain provides a inseminated imago females. Apparently these cessful attempts have been made on 12 oc­ is not necessary as it is for some other may­ constant source of food. have been diverted to the light as they flew casions to hatch H. bilineata eggs partheno- flies such as Ephoron album (Britt, 1962). Large, illuminated reservoir tanks are filled upriver to lay their eggs. genically. Fertile eggs may be obtained H. bilineata eggs hatch in 12 days at 22 C with tap water to which inorganic fertilizer If small amounts of eggs are desired, it is readily, however, by artificial insemination. and in 8 days at 32 C. If the eggs are clumped, is added (20 gm of Armour Vertegreen 10- usually simplest to return the gravid females Eggs stripped from female imagoes or subi­ hatching time is greatly prolonged. The eggs 10-10 per 50 gallons of water) to promote an to the laboratory. They may be transported magoes are mixed with the macerated sexual on the outside of the clumps hatch first be­ algae bloom. This algae-rich water is used easily by picking them up by the wing tips elements of the male in the insect's own body cause they are best oxygenated. to replace water lost through evaporation from and placing them in a large inflated paper fluids, as described by Needham (1935). If the rearing tanks. bag. If egg collection must be postponed for insects arise from the rearing tank in very MAINTENANCE OF NYMPHAL POPULATIONS Eggs may be placed directly into the rearing some reason, the bag and its contained insects small numbers, and if males are not im­ Nymphs have been successfully reared in chamber for incubation or they may be should be refrigerated to slow the metabolism mediately available, females may be kept for galvanized stock-watering tanks, galvanized hatched and then added. In the latter case of the insects and thus lengthen their life up to two days in paper bags in the refrigerator wash tubs, various glass aquaria, and in large it is advisable to place the unopened bags full span. at 10 C. polyethylene bags. The containers are filled of newly-hatched nymphs in the rearing tank Females are dropped, a few at a time, into to a depth of 15 cm with rich garden soil for an hour before opening to equalize the a large water-filled funnel, the stem of which STORAGE AND INCUBATION OF EGGS which is prepared by spading in moderate water temperatures. is fitted with a rubber tube and a pinch Eggs are conveniently stored and incubated amounts of thoroughly composted hay. In­ Nymphal growth is negligible at 14 C, but clamp. As they struggle on the surface of in water-filled polyethylene bags which mea­ organic fertilizer (Armour Vertegreen 10-10- the rate increases with temperature. Only 79 the water, the females release their eggs which sure 10 X 16 X .0015 inches. The eggs are 10) is mixed in at the rate of 100 gm per m2 days were required to develop newly-hatched sink slowly into the stem of the funnel where able to respire because polyethylene is perme­ of soil area. We have successfully used the H. bilineata nymphs to adults in a tank which they may be tapped off in the desired able to oxygen and carbon dioxide. Up to 3 cc same soil for five years by enriching it in was maintained between 24 and 27 C. quantities. of eggs may be stored per bag for 120 days the preceeding manner. When nymphs are crowded, they exhibit Imago females release their eggs most at 12 C with maximum viability (Flattum, Enough water is added to fill the con­ differential growth rates. The larger nymphs readily when they are lying right side up on 1963). After 120 days, the eggs will begin to tainers. Constant aeration is provided to main­ apparently inhibit the growth of smaller the water surface with their outstretched wings hatch at this temperature if oxygen is available tain a high level of dissolved oxygen in the nymphs. In one instance a crowded population adhering to the surface film. They have dif­ to them. water. Polyethylene bags are well suited for which was initiated at about 7000 nymphs ficulty ovipositing unless their wings are im­ We have stored H. bilineata eggs for 380 isolated individuals because the bags are in 2.3 m2 of substrate finally produced 479 mobilized in some manner. nwrron anrl t Vine rpmiirp. nn adult insects. The adults matured at varying lay eggs when they are caugfr Evaporation of water from the roaring tank may cause a deleterious ivever, the last adults emerging 327 or if they are stuck to wet pa1 r the first ones. Differential growth accumulation of dissolved salts in hard-water areas. It may be necessary.

therefore, to drain water from the tank occasionally and to replace it with new water* 410 SHORT PAPERS AND NOTES rates among Hexagenia nymphs has been re­ Erickson, Roger Flattum, Gary Schoening, ported previously by Spieth (1938). Gerrit Kloek, Robert Keller, and Henry Nilsen Nymphs may be collected from large tanks who, as students, helped me in my research. by scooping vigorously, back and forth through the water with a screen-bottomed LITERATURE CITED bucket. This violent agitation dislodges the BRITT, N. WILSON. 1962. Biology of two species nymphs from their burrows and they are of Lake Erie mayflies, Ephoron album (Say) trapped, unharmed in the bucket. This pro­ and Ephemera simulans Walker (Ephemerop- tera). Bull. Ohio Biol. Surv. 1: 1-72. cedure eliminates the tedious task of sifting FLATTUM, ROGER. 1963. Apparent effects of re­ mud to find the nymphs. frigeration on the rate of embryonic develop­ ment of mayfly (Hexagenia limbata) eggs. Nymphs have also been collected by re­ Proc. Minn. Acad. Sci. 31: 82-83. moving the aerator and by scattering granu­ FREMLING, CALVIN R. 1960. Biology of a large lated sucrose at the rate of 500 gm per m2. mayfly, Hexagenia bilineata (Say), of the Upper Mississippi River. Iowa State Univ. Agr. Home The sugar settles to the bottom where it acts Econ. Exp. Sta. Res. Bull. 482: 841-852. as a high biochemical-oxygen-demand pol­ . 1963. A method for determining the dis- lutant. When the oxygen concentration near solved-oxygen concentration near the mud-water interface. Limnol. Oceanog. 8: 363-364. the mud-water interface drops to 3 ppm, the . 1964a. Mayfly distribution indicates wa­ nymphs leave their burrows and rest on the ter quality on the Upper Mississippi River. Sci. mud surface. When the dissolved oxygen 146: 1164-1166. . 1964b. Rhythmic Hexagenia mayfly emer­ concentration drops to 2 ppm (usually in gences and the environmental factors which in­ about 24 hours) the nymphs swim to the fluence them. Verh. Internat. Verein. Limnol. surface of the water where they may be easily 15: 912-916. HOOPES, DAVID T. 1960. Utilization of mayflies collected. They are able to swim for about and caddisflies by some Mississippi River fishes. two days, whereupon they become fatigued Trans. Amer. Fish. Soc. 89: 32-34. and sink to their deaths in the oxygen-deficient HUNT, BURTON P. 1953. The life history and eco­ nomic importance of a burrowing mayfly, zone at the bottom. The bacteria and yeast Hexagenia limbata, in southern Michigan lakes. which decompose the sugar add organic mat­ Mich. Cons. Dept. Bull. Inst. Fish. Res. 4. NEEDHAM, JAMES G., J. R. TRAVER, AND YIN-CHI ter to the soil, thus enriching it for the next Hsu. 1935. The biology of mayflies. Com- population of nymphs. Dissolved oxygen de­ stock Pub. Co., Ithaca, N. Y. terminations at the mud-water interface are SPIETH, HERMAN T. 1938. A method of rearing Hexagenia nymphs (Ephemerida). Ent. News made by siphoning and by using water-filled 49: 29-32. polyethylene bags as samplers (Fremling, CALVIN R. FREMLING 1963). Winona State College Occasionally, a residual population of Winona, Minnesota nymphs is killed so that a tank can be re­ stocked for another experiment. Hot tap wa­ ter (59 C) was added to a tank to kill the nymphs. To our surprise, however, two subimagoes emerged from the tank two days later. Sampling revealed that many nymphs were still alive. They apparently escaped the hot water by burrowing deep into the mud where they remained until the water had cooled. Between rearing experiments, we now routinely add sugar as described previously. A week later, all water is drained from the tank and the soil is stirred thoroughly as hot water is added to insure that each nymph is killed. ACKNOWLEDGMENTS I wish to thank Gerald Nagahashi, Law­ rence Thomforde, Clyde Pasvogel, James