Short-Term Cold Storage of House Fly (Diptera: Muscidae) Embryos: Survival and Quality of Subsequent Stages

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1 PHYSIOLOGY, BIOCHEMISTRY, AND TOXICOLOGY Short-Term Cold Storage of House Fly (Diptera: Muscidae) Embryos: Survival and Quality of Subsequent Stages ROGER A. LEOPOLD Biosciences Research Laboratory, USDA-ARS, Box 5674, Fargo, ND Ann. Entomol. Soc. Am. 93(4): 884Ð889 (2000) ABSTRACT The survival of Musca domestica L. embryos was assessed after storage at 5 C. Chilling tolerance was inßuenced by the length of the storage period and by the age of the embryo at the time of low temperature exposure. Embryos placed into cold storage at 3hofagewere able to survive 3 d with little reduction in larval emergence or adult eclosion and vitality. One-hour-old embryos were the least tolerant of chilling and could not survive1dat5 C. Three patterns of chilling injury were expressed. They are characterized as immediate, accumulative, and latent. Expression of immediate and accumulative injury was linked to the age of the embryo at the time of chilling, and the latent type of injury was expressed during the postembryonic stages of development and was related to length of cold exposure. Exposure of the house ßy embryos to hypoxic conditions did not increase chilling tolerance, and a hyperoxic environment was detrimental to the use of cold storage as a strategy to increase insect shelf life. KEY WORDS Musca domestica, embryo chilling tolerance, chilling injury, hypoxia, hyperoxia, adult emergence DIPTERAN SPECIES ARE the most frequently propagated insects in captivity and are also reared in the highest numbers. Drosophila melanogaster has been widely used in genetic, developmental, and molecular biology studies and the various stocks of this insect maintained around the world is estimated to be 15,000 (Ashburner 1992). The successful eradication of the New World screwworm, Cochliomyia hominivorax (Coquerel), from the United States and Mexico was accomplished by rearing this insect in massive numbers and releasing sexually sterilized ßies to mate with their wild counterparts (Krafsur et al. 1987; Vargas-Teran 1991). This sterile insect technique (SIT) program continues in Central America and the Caribbean and is currently supplied by the USDA/Mexico mass-rearing facility in Tuxtla Gutierrez, Mexico, which has a capacity for producing 300 million ßies per week. Similar SIT programs are being conducted around the world with a number of fruit ßy species (Klassen 1989). During the mass-rearing process, it is beneþcial to be able to hold the eggs needed for propagation of the insects for use at a later date. Short-term storage of eggs obtained from a single oviposition by the core colony would allow insectary managers an opportunity to spread out seeding of subsequent generations over multiple work shifts or weekends. Other beneþts gained from the ability to store eggs include permitting maintenance of a smaller core colony by eliminating waste of eggs, enabling long distance shipping, 1 Mention of a proprietary product does not constitute an endorsement or a recommendation by the USDA for its use. and holding a backup supply of eggs for each production cycle for security purposes. Cold storage of eggs during the rearing process of insects to be released for biocontrol purposes has had limited study, while the low temperature storage of immature parasites within host eggs and storage of host eggs for later use has been extensively investigated (Leopold 1998). Likewise, little study has been conducted on using cold temperatures to store insect eggs for SIT programs, and, for the dipterans, it has been primarily the puparia which have been chilled for stockpiling purposes (Tzanakakis and Stamopoulos 1978, Fabritius 1980, Klunker 1982, Spradberry 1990, Lindquist et al. 1992). Manipulation of the cold storage period to lengthen the time in storage or to ensure that the quality of the insect is not compromised by short-term chilling of the embryo has also received limited attention. Periods of low-oxygen tension have been shown to delay embryogenesis in insects (Ivashchenko 1977). With the black ßy, Simulium ornatum, Meigen precooling at 5 C coupled with low-oxygen tension yielded high survival of embryos subsequently stored at 1 C (Goll et al. 1989). Further, anoxia is often associated with producing or maintaining certain cryoprotective effects that accompany dormancy in insects. Sonobe et al. (1979) showed that in diapausing silkworm eggs there is a reduction of oxygen permeability of the egg membranes which leads to hypoxia, a lowered rate of metabolism, and polyol accumulation. Thus, the aim of this study was to determine survivability of a dipteran livestock pest stored at a subambient, nonfreezing temperature and to assess

2 July 2000 LEOPOLD: COLD STORAGE OF FLY EMBRYOS 885 whether oxygen enhancement or the absence of oxygen is beneþcial to storage. Because insects reared for control purposes have functions beyond reproduction, efforts were also made to determine the vitality of the adults at eclosion after being stored as embryos. The house ßy, Musca domestica L., was used as the insect model for this investigation because it has similarities to many of the dipteran livestock pests. Primarily, the insect shows reduced tolerance to shortterm chilling (Strong-Gunderson and Leopold 1989), embryogenesis is consistent with other dipteran livestock pests in that the house ßy has no demonstrated egg diapause, and it is easily reared in the laboratory. Materials and Methods The eggs were collected by placing 250 ml plastic cups containing larval rearing media covered with paper toweling, which had been moistened with water and a dilute solution of ammonium carbonate, into cages holding 300 of the Orlando Regular strain of house ßies. The ßies were allowed to oviposit for 30 min. Thus, the age of the ßy embryos at the various postoviposition times tested was calculated from the midpoint of the oviposition period. Rearing and maintenance of the colony was as previously described (Leopold 1970). Groups of eggs were placed on moistened paper towels within 250 ml plastic cups and allowed to develop at 28 C for 1, 2, 3, 4, 6, or 9 h before placing them into storage at 5 C. The variation in temperature when the insects were stored at 28 C was 1.0 C and when at 5 C, it was 0.5 C. Before placing them into storage at 5 C, the paper cartons containing the different groups of embryos, were placed into Billups-Rothenberg Modular Chambers (DelMar, CA). These 5-liter capacity, plastic chambers are airtight and have sealable inlet and exhaust ports that allow introduction of ambient air, nitrogen, or oxygen. Eggs were placed into the chambers containing moistened paper towels to keep the humidity close to saturation. The chamber was ßushed with the gas to be tested for several minutes at a delivery pressure of 10 psi and then sealed so that the pressure within the chamber did not exceed 2 psi. Samples of eggs were removed at daily intervals up to 8 d and the particular gas being tested was replaced after each sampling. After removal from the chamber, the eggs were placed into the 28 C humidiþed environment and allowed to hatch into larvae and the survival rate was calculated. Some groups of larvae that were stored at 5 C were placed on the larval growth media, allowed to develop and emerge as adults, and the success of adult emergence was calculated. The reproductive success of the emerged adults was also assayed by separating the sexes before the females reached sexual receptivity and placing them with untreated ßies of the opposite sex. After mating occurred, the individual pairs were separated and the females were placed on oviposition medium and allowed to oviposit their Þrst clutch of eggs. These eggs were placed into plastic petri dishes lined with moist black cloth and the percentage of larvae hatching from Fig. 1. House ßy embryo daily survival after placement at 5 C in ambient air (A) or nitrogen (N) when 1-, 3-, 6-, or 9-h-old (three replicates; 300Ð500 per replicate). the eggs was calculated. The fecundity of individual females was not determined. An attempt was made to assay the Þtness of the adults emerging from the various treatments by placing groups of 20 pupae into the bottom of glass tubes (20 cm diameter, 120 cm long). Sawdust was placed on top of the pupae to a height of 105 cm, which subsequently compacted to cm before the adult ßies began emergence. The sawdust was obtained from untreated pine boards and, before use, was screened with a No. 10, U.S.A. standard testing sieve with 2-mm openings to remove wood chips and obtain a medium of small, uniform wood particles. The sawdust was dried overnight in a 60 C oven and stored between tests in an airtight plastic container. The top of the column was sealed with paraþlm that had been perforated with small holes. Thus, upon eclosion, the adults were forced to crawl through the sawdust to the enclosed space at the top of the column before they could Þnish emergence by expanding and hardening their wings. The number of adults emerging, those adults making it to the top of the sawdust column, and those dying while crawling into the sawdust were recorded. The cold storage experiments in this study were replicated two or three times and within a replicate each treatment group had three individual samples of 75Ð100 insects. The Þtness tests were replicated three times, each treatment group had two samples, and each individual sample size was 20 insects. The results were analyzed by using SigmaStat for Windows (Jandel 1995). After one-way analysis of variance, mean differences between hatching and emergence values were compared by using the Tukey multiple range tests. Results Embryo Survival. The means for the daily survival of embryos aged 1, 3, 6, and 9 h postoviposition, when placed in storage at 5 C in either ambient air or nitrogen for up to 8 d, are shown in Fig. 1. One-hour-old embryos were the most intolerant to cold storage and

3 886 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 93, no. 4 Fig. 2. House ßy embryo daily survival after placement at 5 C when 2-, 3-, or 4-h-old ( SEM, 2 replicates; 300Ð500 per replicate) Fig. 3. House ßy embryo daily survival after placement at 5 C in ambient air or a hyperoxic environment at3hofage ( SEM, 2 replicates; 200Ð300 per replicate) none survived even1dat5 C. As a group, the 3-h-old embryos were the most tolerant to chilling at 5 C. Approximately 75% of the embryos stored in either air or nitrogen survived 4 d cold storage and nearly 50% of the embryos stored in ambient air survived 5 d storage. Storage under nitrogen was beneþcial for only the 6-h-old embryos. Survival was signiþcantly greater (86.2 versus 62.3%, P 0.01) after 3 d storage under nitrogen, but fell to around 20% for both treatments after 4 d. The embryos placed in cold storage at3hof age in ambient air had the best overall survival of all groups tested (P 0.05). To afþrm that the stage of embryonic development at or near the 3-h postoviposition time was the most tolerant to long-term chilling, another test was run where only 2-, 3-, and 4-h-old embryos were placed into storage for up to 8 d in the presence of ambient air (Fig. 2). The 2-h-old embryos had 25% survival after only 1 d ofcold storage and decreased to zero by the fourth day. The 3- and 4-h-old groups had 75% survival up to the fourth day and were statistically similar until the sixth day of cold storage where the 3-h-old group still had 25% of the embryos hatching into larvae and the 4-h-old group was close to zero. On the seventh and eighth day of storage, survival for both the 3- and 4-h-old groups was at or near zero. Comparing ambient air to an oxygen-enhanced environment during cold storage showed that hyperoxia was deterimental. Storing 3-h-old embryos in the presence of a high oxygen concentration decreased survival to the point where on the fourth day only 20% hatching was observed (Fig. 3). In contrast, 75% of the embryos stored in ambient air at 5 C were able to hatch into larvae after four d cold storage. Adult Emergence and Fitness. The effect of embryo cold storage on the rate of the subsequent adult emergence is compared with embryo hatching in Table 1. For the 3-h-old group, the number of adults emerging ranged from 54 to 63% for those embryos stored up to 3dat5 C, whereas control emergence was 64%. However, after4dofstorage, the rate of adult emergence fell to 26%. Adult emergence was essentially down to zero by the sixth day. For the 6-h embryos after 1 d of storage, the adult emergence was about half that of the 3-h groups and by the third day no emergence was observed. Only 10% of the 9-h embryos emerged as adults after only 1 d of storage and emergence was negligible by the second day. Eggs were obtained by male and female reciprocal out crosses from only the 3-h groups stored up to 4 d and from the 6-h groups stored for 1dat5 C. No differences were observed in the rate of embryo hatching from the stored groups when compared with the controls (data not shown). All hatching rates, including the controls, ranged from 76 to 84%. The data shown in Fig. 4 represent eclosion success and a test of the stamina of the emerging adults that had been placed in storage as embryos at 3 h postoviposition for up to 5 d. No death occurred in the column of the adults stored as embryos up to 3dinthecold, but there was 30 and 40% column death after the fourth and Þfth day of storage, respectively. Lack of eclosion is an event late in the ontogeny of the house ßy where an appreciable amount of death occurs a relatively long time after the embryos were removed from storage. Like those adults displaying an inability to traverse the column, adults experiencing 3 dat 5 C as embryos were also at risk for eclosion difþculties. After 5dofcold storage, 45% of the insects failed to ecloseñalmost double the number for the groups stored for 4 d. Discussion For the best possible survival and vitality of house ßy embryos stored at 5 C, the eggs must be placed into cold storage 3 h after oviposition. Hatching, eclosion, the capacity to crawl through a column containing 100 cm of sawdust at eclosion, and subsequent reproduction was relatively unaffected after being placed into the cold for up to 3 d. After4dinstorage, hatching of the 3-h embryos was still 77% of the control but adult eclosion fell to less than half of that of the control. Hatching or eclosion of the other groups (1-, 6-, and 9-h-old embryos) was signiþcantly reduced after only1dofexposure to 5 C. The extreme

4 July 2000 LEOPOLD: COLD STORAGE OF FLY EMBRYOS 887 Table 1. Hatching and adult emergence (mean % SE) from 3-, 6-, and 9-h-old embryos held at 5 C for up to 7 d Storage, days 3 h 1 6h 9h 3h 6h 9h a a a a a a ab ab a ab b b b b b b c c c c d cd d d Ñ Ñ e d e Ñ Ñ f d 0 0 Ñ Ñ Control Age of embryos when placed in storage. Means within columns followed by the same letter are not signiþcantly different, P sensitivity of dipteran embryos to chilling very early in their development has been reported previously, but, except for the study on house ßy cold tolerance of Strong-Gunderson and Leopold (1989), this phenomenon of nonfreezing injury was largely noted in ßies that had been supercooled to temperatures of 10 C or below (Heacox et al. 1985; Myers et al. 1988; Mazur et al. 1992; Leopold 1993, 1998; Miles and Bale 1995). There is good evidence that chilling intolerance of the very young embryos is related to the formation of the blastoderm. At 1hofage, the ßy embryo is a syncytial mass of synchronously dividing cleavage energids embedded in a matrix of yolk particles. Cleavage divisions in house ßy embryos occur at a rapid rate, estimated to be every 8 5 min (West et al. 1968). It was suggested by Strong-Gunderson and Leopold (1989) that the process of cellularization of the blastoderm embryo may be damaged by low temperature exposure, thus resulting in the chilling intolerance. Subsequently, Callaini and Marchini (1989) reported that exposure of Drosophila embryos to 0 C for1h caused formation of abnormal centrosomes at cellularization, and treatment of very young embryos caused arrest of division of cleavage nuclei at metaphase. The signiþcant developmental event(s) occurring for the house ßy embryo are at 3 h for early gastrulation; at 6 h for neuroblast differentiation and ventral/posterior segmentation; and at 9 h for dorsal closure and midgut differentiation (Cantwell 1976). Fig. 4. House ßy emergence success (eclosion and traversing a 100 cm column Þlled with sawdust) after storage as 3-h-old embryos. ( SEM, three replicates; 40 per replicate) (Hatching occurs at 12Ð12.5 h after oviposition when the embryos are held at 28 C.) It is evident that the developmental events occurring at ages tested in this study have different levels of sensitivity to cold exposure. It is not readily apparent what is the cause of this variation in sensitivity, but it may relate to the amount of cellular division or differentiation that is occurring at the time of placement of the embryos into cold temperature. Although death caused by chilling of the 1-h-old embryos is of an immediate type, death of the embryos placed into storage at 3hofageandolder appears to be evoked by an accumulation of damage which eventually becomes lethal at a later embryonic stage. In the case of 3-h-old embryos, there was a rapid decline in hatching after 3 d of storage, and with the 6- and 9-h-old embryos this rate of decline became even more precipitous with an additional day of storage. These shoulder-like survival curves were also observed in an earlier study upon cold storage of house ßy pupae (Leopold et al. 1998). Chilling intolerance of the young pupae and pharate adults in that study was correlated to a higher respiration rate. Individuals having the most tolerance to cold storage had very low respiration rates. Thus, the amount of metabolic activity occurring during a certain developmental stage at the time of low temperature exposure may be a factor that determines the capacity of the embryos to survive a cold storage period. The expression of latent damage appearing in subsequent stages after cold storage of the embryos in the form of reduced adult eclosion and quality is an important observation because it is the adult stage that is the active agent in a control program such as SIT. Rivers et al. (2000) reported that latently expressed death occurred in the pupal or pharate adult stage when the nondiapausing wasp parasitoid, Nasonia vitripennis, was exposed to moderately severe chilling conditions ( 10 C). Death occurred mostly a short time after exposure when the temperature was lower ( 20 C) or when the exposure at 10 C was lengthened. Bucher et al. (1947) observed a similar latent expression of cold-induced injury in the progeny of house ßies that had been exposed as puparia to 6 C for 10 d. The F 1 generation displayed signiþcantly lower hatching, larval survival, and pupation. It was mentioned by Bucher et al (1947) that the reproduction of surviving F 1 adults was normal, but no data were given. This observation coincides with the results of the cur-

5 888 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 93, no. 4 rent study where the hatching of the F 1 generation was also seen to return to control levels. The hypoxic and hyperoxic environments were mostly ineffective in lengthening the time for survival for the embryos stored at low temperature, unlike what was shown to be beneþcial for the blackßy, S. ornatum (Ivashchencko 1977, Goll et al. 1989). Hypoxia typically causes a cessation or lowering of the metabolic rate (Hochachka and Guppy 1987). In theory, this cessation would be in addition to slowing of metabolic processes by exposure to 5 C. However, the elimination of ambient air via placement into a strictly nitrogen environment gave no overall protective effect except for the 6-h-old embryos and that effect dissipated after 1 d. It is quite possible that the ßy embryos do not contain the biosynthetic mechanisms, such as the ability to alter the source of substrate, to change enzyme concentration and kind, and to modify membrane and organelle structure, needed to gain the beneþts of low oxygen tension. Cold storage under a hyperoxic environment was detrimental to survival of the 3-h embryos. Oxygen toxicity caused by prolonged exposure to O 2 at partial pressures above that found in ambient air is not uncommon and may be either caused by direct involvement in the biological injury or acting as a precursor to formation of potent oxidizing agents (Jones 1985). Miquel et al. (1975) and Baret et al. (1994) demonstrated that continuous exposure of a highly oxygenated atmosphere to D. melanogater adults decreased their life span. In the latter study, histological examination of ßies exposed to high oxygen concentrations suggested that membrane damage probably caused by lipid peroxidation was the source of the lower viability. The Þndings in this study emphasize the importance of determining the most chill tolerant stage of development when using cold storage as a means to increase the shelf-life of insects and, in this case, it is the 3-h-old embryo. Further, as chilling injury can be incurred when the period of storage is too long, it is also important to determine whether any latent harmful effects arise in subsequent stages. Finally, a method for estimating vitality at eclosion after cold storage was developed. Should this method have correlation to determination of ßight ability, mating propensity, or dispersal adequacy, it may well prove to be a valuable tool in assessing the total quality of dipteran insects reared for release in control programs. Acknowledgments I thank Janice OÕConnor for assistance in conducting these studies and special thanks go to Thomas Freeman and Peter Atkinson for their helpful comments on the manuscript. References Cited Ashburner, M Frosted ßies. Science (Washington, DC) 258: 1896Ð1897. Baret, P, A. Fouarge, P. Bullens, and F. A. Lints Lifespan of Drosophila melanogater in highly oxygenated atmospheres. Mech. Aging Dev. 76: 25Ð31. Bucher, G. E., J. W. Mac, B. Cameron, and A. Wilkes Studies on the house ßy (Musca domestica L) II. The effects of low temperature on laboratory-reared puparia. Can. J. Res. Sec. D Zool. Sci. 26: 26Ð56. Callaini, G., and D. Marchini Abnormal centrosomes in cold-treated Drosophila embryos. Exp. Cell. Res. 184: 367Ð374. Cantwell, G. E Embryonic and postembryonic development of the house ßy (Musca domestica L.). U.S. Dep. Agric. Tech. Bull Fabritius, K Storage of pupae of Musca domestica L. parasitised by Muscidifurax raptor Gir. & Sand. (Hymenoptera: Chalcidoidea). Stud. 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