ISSN 0013-8738, Entomological Review, 2009, Vol. 89, No. 9, pp. 1019–1024. © Pleiades Publishing, Inc., 2009. Original Russian Text © E.B. Vinogradova, 2009, published in Entomologicheskoe Obozrenie, 2009, Vol. 88, No. 4, pp. 512–520.

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Methods of Short-term Storage of Cultures of the Blowfly Calliphora vicina R.-D. (Diptera, Calliphoridae)

E. B. Vinogradova Zoological Institute, Russian Academy of Sciences, St. Petersburg, 199034 Russia

Received February 10, 2009

Abstract—Three methods of short-term storage of the blowfly Calliphora vicina strains are considered based on the experimental study of 21 strains originating from different parts of the species range. The colony can be pre-served as diapausing adults at 6° and darkness for 2–3 months or more, depending on the geographical origin of the population. During the first five days of adult life the flies should be kept at 12° and short day on a sugar diet, after which they should be transferred into a refrigerator. During artificial hibernation the flies also require periodic sugar feeding every 20 days (3–4 h at 20°C) to maintain their vital functions. The combination of temperatures of 20–23°C and a protein diet terminates reproductive diapause, and oviposition starts in 10–17 days. The fly strain may be preserved as reproductive females at 6°C and darkness with sugar feeding. Flies also require periodic sugar feeding at 20°C (3–4 hours). In this case the flies start laying eggs 2–3 days after being transferred to 20–23°C. The preservation of diapausing larvae is a more reliable method of prolonged strain storage. In this case the flies of ma-ternal generation are maintained at 20–23°C on sugar and protein diet. The egg rafts laid during 5–6 hours are then transferred into 12°C and short day until hatchment. The hatched larvae should be immediately placed into a refrig-erator (2–3 or 5–6°C), where they feed during 1–1.5 months and enter diapause. For strain restoration, the diapaus-ing larvae should be transferred into 20–25°C, where they pupariate in 3–5 days and the flies emerge in nearly 10 days.

DOI: 10.1134/S0013873809090012

The blowfly Calliphora vicina has long been used as a model object in a broad range of researches, ow-ing to its large size, easy culturing, and the availability of detailed descriptions of internal and external mor-phology of all the development stages (Lowne, 1890–1895). This species was used in physiological studies of all the organ systems of insects, especially in the fields of endocrinology, sensory physiology, and flight energetics (Vinogradova, 1984). The larvae of the blowfly were recently shown to have curative proper-ties. Biologically active peptides termed alloferons, isolated from the blowfly larvae, were found to in-crease viral immunity; a preparation for treatment of herpes and papillomas, based on these peptides, is manufactured under the trademark Alloidin (Chernysh, 2006). The blowfly has a broad Holarctic distribution and represents an obligatory component of the synan-thropic dipteran complex in the temperate and sub-tropical zones. Its seasonal adaptations, adult (repro-ductive) and larval diapauses, and their photothermal control are rather well studied (Vinogradova, 1984, 1991). The reproductive diapause occurs in all the 21 examined populations, originating from different parts

of the species distribution range, from 69 to 38°N and from 10 to 85°E. The main role in diapause induction belongs to temperature, while photoperiodism is of secondary importance. However, photoperiodism par-ticipates in development of the maternal effect: the short day acting upon the maternal organism deter-mines a more pronounced tendency to larval diapause in the progeny, whereas exposure of the maternal or-ganism to the long day results in non-diapause devel-opment of the progeny. The maternal effect can be realized within a certain range of temperatures (below 14°C) at which the larvae develop. Besides the eco-logical factors, diapause formation is affected by in-ternal factors, such as the age of the female (Nesin et al., 1995) and endogenous processes in a sequence of generations developing under constant conditions (Vinogradova and Reznik, 1999, 2002).

Since the blowfly is broadly used as a laboratory object, it would be very convenient to develop a tech-nique for short-term storage of its culture. This com-munication considers various methods of preservation of Calliphora vicina strains at different stages of de-velopment.

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MATERIALS AND METHODS

This paper presents the experimental data obtained in 2007–2008 and earlier. In all, we studied the cul-tures of the blowfly originating from 21 localities from different parts of its distribution range. Although the technique for blowfly cultivation was earlier described (Zinov’eva and Vinogradova, 1972), some parameters related to temporary storage of the culture will be considered in greater detail. To obtain diapaus-ing adults, freshly emerged individuals were first kept for 5 days at 12-h photoperiod and 20°C and fed on carbohydrates (sugar) to build up nutrient reserves, and then transferred into the refrigerator (6°C, dark-ness). The physiological state of the females was assessed by periodic dissection of 25–40 individuals; the females with follicles at I stage were regarded as diapausing.

The diapausing larvae were obtained from the flies that were kept under short-day conditions (12 L) at 20°C. The flies readily laid eggs on fish, liver, and similar food placed on humid filter paper. The group layings (egg rafts) accumulated in 5–6 h were main-tained under short-day conditions at 12°C until hatch-ment; the freshly hatched larvae were, when neces-sary, transferred into low-temperature conditions (6 or 2–3°C), because even a short exposure to 12°C and especially to 20°C significantly decreased the fraction of diapausing larvae. At low temperatures the larvae developed to the diapausing stage. During the first 1–1.5 months the larvae were irregularly fed on pro-teinaceous food (fish, meat, liver, etc.). Slightly mois-tened sawdust was used as substrate for the larvae. If the sawdust became too humid due to feeding of the larvae and decomposition of the food substrate, the larvae sometimes tried to crawl out of the container; in this case, a portion of dry sawdust was added. After the larvae ceased feeding and evacuated their digestive tracts, they were transferred into clean sawdust for storage; the substrate was periodically moistened when it became too dry. The larvae in our experiments were kept in 0.5-l or 1-l glass jars covered with dense cloth; containers of greater volume may be used to obtain large quantities of diapausing larvae.

To study the dynamics of cold reactivation of the diapausing blowfly larvae, 4–5 samples of 100 larvae each were transferred at different dates from 2–3°C to 12°C (in the dark), and the number of puparia formed was determined every day.

The statistical data processing to determine the time of pupariation included determination of the median and the upper and lower quartiles.

RESULTS AND DISCUSSION

The experimental data on seasonal adaptations of the blowfly suggest three different methods of storage (short-term conservation) of the culture of this fly, each method having specific advantages and drawbacks. They are considered in greater detail be-low.

Storage of Diapausing Females The principal role in induction of the reproductive

diapause in the blowfly belongs to the temperature, whereas the effect of the photoperiod is less signifi-cant. A decrease in temperature from 20 to 12 and 6°C markedly increases the fraction of diapausing indi-viduals. For example, in blowfly cultures originating from Murmansk and St. Petersburg and kept at 12-h light regime and 12°C, about 60% of the females enter diapause on the 20–30th day after emergence. Their diapause ceases by the 50–70th day owing to sponta-neous reactivation (Vinogradova, 1988). The inci-dence of diapause under these conditions varies among different geographic forms of the blowfly and also changes in the sequence of generations of a continuous culture as a result of endogenous processes (Vinogra-dova, 1991; Vinogradova and Reznik, 1999, 2002). The strains of different geographic origin differ in the range and the total degree of variability of the fraction of diapausing females. For example, the strains from St. Petersburg, Moscow, Dnepropetrovsk, and Ash-gabat on the 30th day in the 4th generation contained 35, 4, 16, and 4% of diapausing females, and in the 5th generation, 55, 10, 12, and 10%, respectively. In blow-flies from St. Petersburg and Ashgabat the fraction of diapausing individuals varied from 0 to 56% over the 9–11th consecutive generations; the remaining strains showed a smaller range of variation, from 0 to 16%. The temperature of 6°C in the dark induced diapause in all the flies. The data for 10 geographic forms are shown in Table 1. All the flies entered diapause at the age of 30 days, but the rates of their subsequent spon-taneous reactivation varied. The diapause could be terminated after 2 to 6 months. A relatively strong diapause was observed not only in the strains from the northern and middle parts of the distribution range (Murmansk, St. Petersburg, Moscow) but also in those from more southern areas (Ashgabat, Sukhumi). Thus,

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there was no direct relationship between the strength of reproductive diapause and geographic origin of the culture.

Contrary to what is observed in other fly species, the reproductive diapause in the blowfly is not accom-panied by extensive hypertrophy of the fat body. At 6°C, from 22 to 82% of individuals are characterized by the 1st (initial) stage of fat accumulation and there-fore require systematic carbohydrate and protein diet. The dissections showed that all the flies fed on carbo-hydrates, and 21–65% of them also consumed pro-teins. As a result of long exposure to low tempera-tures, some of the flies get reactivated and the others die. Noteworthy that some of the reactivated females may produce eggs even at 6°C.

Reactivation of diapausing flies could be induced at any moment by raising the temperature. In particular, diapause in all the flies is terminated within 30 days after they have been transferred from 6°C to 12°C and photoperiod 12L, and within 10–17 days if they are transferred to 20°C (Vinogradova, 1989).

Thus, the blowfly culture can be stored for 2–3 months (and some strains even for 5–6 months) in the form of diapausing females kept in the dark at 6°C. During the first 5 days of adult life, the flies should be kept at 12L photoperiod and 12°C on a car-bohydrate diet to allow them to accumulate the mini-mum nutrient reserves. Later, they should be taken from the refrigerator every 20 days and allowed to feed on proteins and carbohydrates for 3–4 h at 20°C. The culture can be reactivated by raising the tempera-ture to 20°C.

Storage of Reproductively Active Females

It was experimentally shown that egg resorption and inactivation of ovipositing females could not be in-duced by any photothermal conditions (Vinogradova, 1989). If the flies which have started ovipositing at 20 or 12°C are transferred into low temperature condi-tions (6°C), they do not stop feeding and may lay eggs periodically for 46–80 days. Although this method provides shorter periods of storage, it allows the cul-ture to be reactivated very quickly should the need arise, because the flies in this state start ovipositing 2–3 days after being transferred into long day condi-tions at 20–23°C.

Storage of Diapausing Larvae

The larval diapause in the blowfly is controlled by the state of the maternal organism, and the principal factor regulating the maternal effect is photoperio-dism. For example, in the strain originating from Be-lomorsk the fraction of diapausing larvae at 12L and 12°C on the 45th day after hatching decreases in rela-tion to the photoperiodic regime affecting the females: it comprises 100, 82, 19, and 4% at photophases of 12, 16, 17, and 20 h (Vinogradova, 1991). To obtain the maximum number of diapausing larvae, the flies of the maternal generation should be kept under short day conditions; the recommended day length varies de-pending on the geographic origin of the material and maybe determined experimentally. In most cases, 12-h photoperiod can be used. It should be noted that pho-toperiodism mediated by the maternal effect deter-mines not only the fraction of diapausing larvae but also the properties of diapause itself, shorter photo-

Table 1. Dynamics of the fraction of diapausing females of the blowfly (%) at 6°C in the dark, based on the data of E.B. Vinogradova (1989, 1997)

Days Localities and coordinates

30 60 90 120 150 180 Murmansk, Russia, 69°N, 39°E 95 95 90 90 51 45 St. Petersburg, Russia, 60°N, 30°E 97 70 44 19 7 – Moscow, Russia, 56°N, 38°E 100 100 54 18 2 Dnepropetrovsk, Ukraine, 48°N, 36°E 100 100 57 43 – Yalta, Ukraine, 44°N, 34°E 100 47 35 8 Sukhumi, Georgia, 43°N, 41°E 100 100 44 42 2 Aktyuz, Tajikistan, 43°N, 76°E 100 48 35 23 10 0 Shafirkan, Uzbekistan, 40°N, 64°E 98 81 56 40 – Dushanbe, Tajikistan, 39°N, 69°E – 44 9 4 Ashgabat, Turkmenistan, 38°N, 58°E 100 100 100 95 95 56

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periods inducing a stronger diapause (Saunders, 1987; Vinogradova, 1991).

Some of the larvae produced by short-day flies enter diapause already at 12°C; however, diapause is more efficiently induced by such temperatures as 5–6°C and even 2–4°C, i.e., very close to the development threshold (Vinogradova, 1991). In this case photoperi-odism has no effect at all, and the larvae can be kept in the dark. The duration of storage of diapausing larvae is determined by the time of their pupariation as a result of spontaneous reactivation at the given tem-perature. The data on pupariation of the larvae that developed at 6 and 12°C are given in Table 2. They are represented as median values, i.e., time (days) needed for pupariation of 50% of the larvae. The dif-ferent geographic forms vary with respect to this pa-rameter. Of the 13 strains listed in the table, the me-dian value of 40–50 days was observed only in the two southernmost populations (Ashgabat and Kulyab); in three other strains (Rybachy, Moscow, and Yalta) the median value was 80–99 days, and in the remaining strains it varies from 111 to 190 days. The other half of the larvae takes even longer to reactivate, which means that it can be stored for over 6 months. Strains of the latter group can be most conveniently stored in the form of diapausing larvae.

The diapausing larvae may also be reared at a lower temperature of 2–3°C. In this case, the period of their feeding and development to the diapausing stage in-creases to about 30 days but the possible time of storage also noticeably increases, to 8–9 and even 12 months. This parameter certainly depends on the geographic origin of the material. Although occasional puparia may appear at the end of the storage period, mass pupariation is usually not observed because the temperature is too low for morphogenesis.

The culture activation can be achieved by raising the temperature to 12, 20, or 25°C. The rates of pu-pariation of the larvae that have developed at 6°C are shown in Table 3. Three geographic forms from the northern, middle, and southern parts of the range (Murmansk, Tomsk, and Shafirkan) differ in their rates of pupariation both at 6 and at 12°C. The larvae of the southern form pupariate much faster than those of the other two forms. For example, in the regime “6→12°C”, 95% puparia of the southern form appear within 20 days, whereas in the other two forms a simi-lar result (75–80%) can be achieved only within 100–120 days. In the regime “6→20°C” pupariation is accomplished in mere 5 days, except for the strain from Murmansk, in which 35% of the larvae pupariate only after 10 days, and 77%, within 11–28 days. The greatest activation potential is observed in the regime “6→25°C”; at this temperature pupariation in all the three forms proceeds very rapidly, reaching 78–84% within the first 3 days, and 95–100%, by the 5th day. The geographic differences in the dynamics of pu-pariation, evident at 12 and 20°C, are leveled at 25°C. At the initial temperature of 6°C, puparia start to ap-pear in small numbers at the 60–80th day, and the entire process takes up to 200 days.

The dynamics of cold reactivation of the larvae was studied in special experiments with strains from Ham-burg (53°N, 10°E, Germany), Kobuleti (42°N, 41°E, Georgia), Tomsk (56°N, 85°E), and “69 km Flag Sta-tion” (60°N, 30°E, Leningrad Prov.). The larvae at the age of 5 to 10 months, kept for different periods of time in the state of diapause at 2–3°C, were transferred to 12°C in the dark (Fig. 1). In spite of the low preced-ing temperature, the larvae pupariated quickly. The median value for all the four strains varied from 4 to 9 days but in most cases (8 out of 14), it was 4–6 days. The variability of this parameter within each strain may be determined by certain heterogeneity of fly generations (the experiments were carried out in dif-ferent years). No significant difference was observed

Table 2. Median values reflecting pupariation of the larvae of Calliphora vicina developing under different photother-mal conditions, based on the data of E.B. Vinogradova (1991)

Median, days Localities and coordinates 12 h,

12°C darkness,

6°C St. Petersburg, Russia, 60°N, 30°E 100 190 Rybachy, Russia, 56°N, 20°E 69 90 Moscow, Russia, 56°N, 38°E 77 80 Belaya Tserkov, Ukraine, 50°N, 30°E 110 149 Dnepropetrovsk, Ukraine, 48°N, 36°E 52 111 Yalta, Ukraine, 44°N, 34°E 22 99 Sukhumi, Georgia, 43°N, 41°E 100 163 Aktyuz, Tajikistan, 43°N, 76°E 99 190 Baku, Azerbaijan, 40°N, 50°E 20 131 Yerevan, Armenia, 40°N, 45°E 20 120 Kondara, Tajikistan, 39°N, 69°E 40 135 Ashgabat, Turkmenistan, 38°N, 58°E 18 50 Kulyab, Tajikistan, 38°N, 70°E 16 40

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between the different geographic forms. It should be noted that the batches of 7-month-old larvae were characterized by the highest median values in all the cases; however, this is likely to have been caused by some common factor, probably a somewhat lower final temperature. The data obtained indicate that the larvae diapausing at 2–3°C reactivate rather quickly; 5–6 months are sufficient at least for the

above strains. The subsequent pupariation is blocked by low temperature but can be accomplished very fast if the larvae are transferred into a higher tempera-ture.

Thus, the blowfly culture stored as diapausing lar-vae at low temperatures can be easily reactivated by raising the temperature. At the final temperature

Table 3. Pupariation (%) of diapausing larvae of Calliphora vicina of different origin in constant (6°C) and changing tem-perature regimes

Regime: 6 → 12° Origin

20 40 60 80 100 120 Murmansk 20 22 30 40 49 80 Tomsk 32 45 62 68 75 Shafirkan 95 Regime: 6 → 20°C 1 2 3 5 9 10 28 Murmansk 0 0 0 0 0 35 77 Tomsk 0 46 77 94 100 Shafirkan 2 80 84 100 Regime: 6 → 25°C 1 2 3 4 5 Murmansk 0 59 82 90 95 Tomsk 0 60 78 90 96 Shafirkan 0 12 84 100 Regime: 6°C constant 40 60 80 100 140 180 200 Murmansk 0 0 11 25 45 55 83 Tomsk 0 0 18 54 67 76 85 Shafirkan 0 10 16 84 100

Note: The days are shown in italics.

Pupariation of diapausing larvae of Calliphora vicina developing in the dark at 2–4°C, in response to transfer into 12°C in the dark. Abscissa: duration of exposure to low temperature (months) and the place of origin of the strain; ordinate: medians and quartiles (days). The experiments with 7 and 10 months of exposure to low temperature were carried out in 2007, those with 5 and 6 months, in 2008.

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of 20–25°C, adult flies emerge from puparia in about 10 days.

CONCLUSIONS Based on extensive experimental data obtained in

21 strains of C. vicina from different parts of its distri-bution range, three methods of short-term storage of the blowfly culture can be proposed.

The blowfly culture can be stored in the form of diapausing females kept in the dark at 6°C, for 2–3 months or more, depending on the geographical origin of the population. During the first 5 days of adult life the flies should be kept at 12°C and a 12-h photoperiod on a carbohydrate diet and then transferred into a low temperature. To maintain the vital functions of the flies during artificial hibernation, they should be allowed to feed on carbohydrates for 3–4 h every 20 days at 20°C. The diapausing flies can be reactivated at any moment by transferring them to 20°C, under which conditions they will start oviposit-ing in 10–17 days.

The culture can also be stored in the form of active ovipositing flies. In this case, the flies are kept at 20–23°C and a long day photoperiod on a carbohy-drate and protein diet until they start ovipositing, after which they are transferred into 6°C in the dark. They also require periodic feeding at 20°C. The flies can be stored in this condition for up to 1–2.5 months; they resume oviposition 2–3 days after being transferred to 20°C.

For the culture to be stored in the form of dia-pausing larvae, it should be prepared in a sequence of stages: keeping the maternal generation of flies at a short day and 20°C on carbohydrate and pro- teinaceous substrates; collection of the group layings produced within 5–6 h; transfer of these eggs to a 12-h photoperiod and 12°C until hatchment; and transfer of freshly hatched larvae to a low temperature (5–6 or 2–3°C). The larvae feed and reach the diapausing stage, in which they can be stored for 8–9 and even 12 months (depending on their geo-graphic origin and the temperature). The culture can be reactivated by temperatures of 20–25°C; the larvae pupariate in 3–5 days and the adults emerge in 8–10 days.

REFERENCES

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10. Vinogradova, E.B. and Reznik, S.Ya., “Endogenous Changes of the Tendency to Diapause in the Blowfly, Calliphora vicina (Diptera, Calliphoridae),” Proc. Zool. Inst. RAS 281, 151–155 (1999).

11. Vinogradova, E.B. and Reznik, S.Ya., “Endogenous Changes of the Tendency to Larval Diapause in Labora-tory Generations Sequences of the Blowfly, Calliphora vicina R.-D. (Diptera, Calliphoridae),” Int. J. Dipterol. Res. 11 (1), 3–8 (2002).

12. Zinov’eva, K.B. and Vinogradova, E.B., “Regulation of Seasonal Development of Blowfly Parasites. Ecological Regulation of Winter Adaptations in Calliphora vicina R.-D. (Diptera, Calliphoridae),” in Host-Parasite Inter-actions in Insects (Leningrad, 1972), pp. 90–99 [in Rus-sian].