Tetanops Myopaeformis) Larvae in Cold Storage

ARTICLE IN PRESS

Journal of Insect Physiology 54 (2008) 691–699 www.elsevier.com/locate/jinsphys

Multi-year survival of sugarbeet root maggot (Tetanops myopaeformis) larvae in cold storage

Anitha Chirumamillaa,Ã, George D. Yocumb, Mark A. Boetela, Robert J. Dregsetha

aDepartment of Entomology, North Dakota State University, Fargo, ND 58105, USA bInsect Genetics and Biochemistry Research Unit, RRVARC-USDA-ARS, Fargo, ND 58105, USA

Received 27 August 2007; received in revised form 22 January 2008; accepted 23 January 2008

Abstract

To test the hypothesis that long-term survival of sugarbeet root maggot in storage is facilitated by larvae undergoing prolonged diapause, respiration and gene expression patterns of ﬁeld-collected diapausing larvae were compared with those of 1-, 2-, and 5-year laboratory-stored larvae. Additional assessments were made on post-storage survival, emergence, and reproductive ﬁtness of stored larvae. Respirometry, carried out at 5 and 20 1C revealed no differences among respiration rates of initially diapausing and long-term stored larvae. A 151 increase in temperature elevated respiration in both diapausing and stored larvae, with levels of CO2 release ranging between 8- and 14-fold higher at 20 1C than at 5 1C. Similarly, 6–10-fold increases in O2 consumption levels were observed at the higher temperature. A transcript with sequence similarity to the fat body protein 2 (Fbp2) gene was highly expressed in diapausing larvae, and trace levels were expressed in some samples of 1-year stored larvae. However, no expression was detected in 2- and 5-year stored larvae. Survival and emergence studies of stored larvae revealed mixed populations of diapausing (i.e., the 5–17% of larvae that did not pupate) and post-diapausing (62–84% of larvae pupated) insects, with a high incidence of pupation (62%) and emergence (47%), even after 4 years in cold storage. Therefore, extended survival of Tetanops myopaeformis larvae in long-term cold storage is facilitated by two mechanisms, with a majority of larvae in post-diapause quiescence and a smaller fraction in a state of prolonged diapause. r 2008 Elsevier Ltd. All rights reserved.

Keywords: Diapause; Post-diapause quiescence; Respirometry; Sugarbeet root maggot; Fat body protein 2; Long-term storage survival

1. Introduction percent of a population can remain in diapause more than 1 year to survive year-to-year variability; however, the Insects are able to overcome the challenges of changing mode of manifestation and conditions for its induction and seasons and associated cyclic ﬂuctuations in environmental termination are not fully understood. Studies by Soula and conditions by undergoing a genetically programmed, Menu (2005) revealed that the extended life cycle of the dynamic state of low metabolic activity known as diapause chestnut weevil, Curculio elephas Gyllenhal, was the result (Tauber et al., 1986). Insects also face unpredictable of prolonged diapause occurring secondarily to a develop- growing seasons that vary in length and suitability for mental phase. This was in contrast to the usual hypothesis growth and development (Hanski, 1988). Diapause can of extended, uninterrupted normal winter diapause pre- also serve as a potential adaptation to this type of seasonal viously described in the Colorado potato beetle, Leptino- variability in the form of extended diapause, which is tarsa decemlineata Say (Ushatinskaya, 1984). An synonymously referred to as superdiapause (Ushatinskaya, additional form of dormancy, referred to as post-diapause 1984), prolonged diapause (Tauber et al., 1986), and extra- quiescence, involves insects remaining in a dormant state long diapause (Hanski, 1988). The adaptive biological beyond the typical duration of normal diapause due to the phenomenon underlying all of these terms is that a small absence of favorable environmental conditions. Although no morphological differences are known to exist between ÃCorresponding author. Tel.: +1 701 239 1475; fax: +1 701 239 1348. diapause and post-diapause quiescent insects, there lies a E-mail address: [email protected] (A. Chirumamilla). ﬁne line of difference in that the former are regulated

0022-1910/$ - see front matter r 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2008.01.008 ARTICLE IN PRESS 692 A. Chirumamilla et al. / Journal of Insect Physiology 54 (2008) 691–699 endogenously while the latter are controlled exogenously pared to those that are non-diapausing (Denlinger, 2002). (Tauber et al., 1986; Danks, 1987; Hodek, 1996, 2002; Transition from diapause to post-diapause development, Kostal, 2006). marked by a sudden shift in the expression pattern of The sugarbeet root maggot, Tetanops myopaeformis diapause-regulated genes, was observed in the flesh fly, (Ro¨der) (Diptera: Ulidiidae), is a major economic insect Sarcophaga crassipalpis Macquart when studied under pest of sugarbeet in the Red River Valley of Minnesota and laboratory conditions at 20 1C(Yocum et al., 1998; North Dakota, and it is also a problem for growers in the Rinehart and Denlinger, 2000; Rinehart et al., 2000, western United States. Adult flies begin emerging during 2001). Recent findings demonstrate that the thermal May and June, and females lay most eggs next to sugarbeet history of an insect has a major influence on the gene seedlings in June. Eggs hatch within 5–7 days, and larvae expression pattern during its transition between diapause promptly begin feeding on roots of sugarbeet seedlings. and post-diapause development (Hayward et al., 2005; Feeding continues as larvae develop through three instars. Yocum et al., 2005, 2006). Larvae cease feeding after reaching full size (i.e., late July A better understanding of the physiological status and into September), and initiate diapause that lasts at least 6 mechanisms that enable prolonged survival of T. myopae- months. Larvae overwinter in the soil at depths of 5–35 cm, formis larvae in storage could provide insight regarding and move to within 10 cm of the soil surface for pupation, the phenology and field physiology of sugarbeet root in late March or early April (Callenbach et al., 1957; maggot. In this study, we test the hypothesis that multi- Harper, 1962; Whitfield, 1984; Anderson, 1986; Bechinski year survival in T. myopaeformis larvae was facilitated et al., 1989, 1990). Studies conducted by Whitfield (1984) by larvae maintaining a state of diapause throughout and Whitfield and Grace (1985) indicated that sugarbeet long-term (i.e., 1, 2, 3, 4, and 5 years) periods of cold root maggots are freeze tolerant and univoltine. Observa- storage. tions by previous investigators suggest that T. myopaeformis undergoes an obligate diapause that requires chilling 2. Materials and methods up to 6 months for termination (Callenbach et al., 1957; Harper, 1962; Klostermeyer, 1973). 2.1. Insect sampling The ability of sugarbeet root maggots to survive prolonged (e.g., 51 months) periods in cold storage (5 1C) Experiments were carried out on different life stages (i.e., has been reported earlier by Kruger (1986), while the second instars, diapausing third instars, post-diapause current data shows that the third-instar larvae collected in third instars, pupae, and adults) of T. myopaeformis the months of August and September can be successfully collected from the field during the year of experimentation maintained alive in cold storage (671 1C) for up to 6 years and on matured third instars that had been collected and (M.A.B., unpublished data). Based on the time of maintained in laboratory storage, at 671 1C for 1, 2, 3, 4, collection, it has been suggested by previous authors that or 5 years. Larvae for emergence studies were collected those larvae are in diapause before entering the storage from multiple fields in different years (1973–1985) near St. (Callenbach et al., 1957; Harper, 1962; Klostermeyer, 1973; Thomas, North Dakota, during July through September. Anderson, 1986); however, it was not known whether the All insects used for respiration and gene expression studies larvae were maintaining the same physiological state were collected from fields located in Pembina County, throughout long-term storage. Besides, there were no North Dakota. Different developmental stages used for previous reports on incidence of prolonged dipause in respiration studies were collected during the field season of sugarbeet root maggot populations, either in the labora- 2004, whereas the 1-, 2-, and 5-year stored larvae used in tory or in field conditions. Prolonged diapause, resulting respiration and gene expression studies were collected from either a simple extension of normal winter diapause during August in 2003, 2002, and 1999, respectively. or larvae re-entering a second diapause following the Larvae and pupae were dug from the soil, placed into winter diapause (Soula and Menu, 2005), could be allowing plastic bags of loose field soil. The bags were transferred to this phenomenon to occur. Alternatively, the larvae could a plastic cooler and transported to the laboratory where be in a state of post-diapause quiescence (Tauber et al., they were washed and transferred into clean plastic storage 1986; Hodek, 1996) and waiting for the return of favorable bags filled with moist silica sand. Insects in bags were then temperatures. stored in an incubator at 671 1C. Larvae that had been The collective characteristics of diapausing individuals, cold-stored were either in the pre-diapause phase (July often referred to as diapause syndrome, include several collected) at the time of collection and had entered complex physiological and behavioral traits such as diapause soon after collection, or were already in diapause uniquely low metabolic activity associated with reduced (August/September collected). respiratory rate, relative inability to respond to environ- Adult flies for the experiments were collected from mental cues, increased resistance to environmental ex- wooden utility poles during high periods of activity and tremes, and altered or reduced behavioral activity (Tauber placed in jars with a cotton plug soaked with honey and et al., 1986; Danks, 1987). In addition, diapausing water. These experiments were carried out to develop a individuals exhibit specific gene expression patterns com- better understanding of various physiological phenomena ARTICLE IN PRESS A. Chirumamilla et al. / Journal of Insect Physiology 54 (2008) 691–699 693 associated with diapause and long-term storage. Three The number of larvae in each batch ranged from 49 to independent experiments involving respirometry, differen- 1215. During the months of May and June, the larvae were tial gene expression evaluations, and assessments of taken out of the incubator and maintained at 12L:12D, post-storage survival, emergence, and reproductive perfor- 25 1C, and 75–80% relative humidity (first sampling) to mance were conducted on T. myopaeformis larvae. assess percent pupation, percent emergence, sex ratio, Metabolic rates of all post-egg stages were evaluated at fecundity, and percent egg hatch. Observations were 20 1C. Additional studies were done at 5 and 20 1Cto continued for 90 days to quantify percent pupation. Larvae compare the field-collected overwintering larvae with those that did not pupate (NP) but remained alive were returned that had been stored for extended periods in the to the incubator for storage and observations were laboratory. Larval responses to temperature increases were repeated the following year (second sampling). assessed to determine if prolonged diapause could explain the ability of larvae to survive long-term laboratory cold 2.4. Differential gene expression storage. 2.4.1. RNA extraction and differential display 2.2. Respirometry Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used according to manufacturer’s protocol to extract total An automatic respirometer (Sable Systems, Henderson, RNA from field-collected diapausing (November 2004) NV, USA), programmed to operate at a flow rate of larvae, and those stored in the laboratory for 1, 2, and 5 100 ml/min, was used for measuring oxygen consumption years. Differential display was carried out according to the and carbon dioxide production of test insects. Measure- manufacturer’s protocol by using GeneFishingTM DEG ments were taken employing constant-volume respirometry Premix kits (Seegene, Rockville, MD, USA). Fragments (Lighton, 1991). Insects were acclimatized to the respective were cloned and sequenced, and primers were designed temperatures for 2–3 h before measurements, and both to synthesize probes using a DIG labeling kit (Roche oxygen and carbon dioxide concentrations were measured Applied Science, Indianapolis, IN, USA). The BlastX on five to seven individual insects of each selected stage at program (Altschul et al., 1997) was used to search the each temperature. Measurement intervals ranged from 1 to GenBank sequence repository and establish sequence 3 h for non-diapausing stages and 3 h to 3 days for identity. The nucleotide sequence for clone D20A2b was diapausing and stored larvae. In a comparative study deposited in GenBank and assigned accession number between field-collected diapausing and laboratory-stored EV413920. larvae, respirometry was carried out at two different temperatures. First, the readings were taken at 5 1C, and 2.4.2. Northern blot analysis later the same larvae were subsequently maintained at Concentrations of RNA samples were determined using 20 1C to measure responses to an increase in temperature. a spectrophotometer, and ethidium bromide was added to Data were collected using the Sable Systems Data all samples to ensure equivalent loading. Total RNA (5 mg/ Acquisition Program (http://www.sablesys.com/datacan. sample) was separated on a 1% agarose denaturing gel html) in accordance with manufacturer’s protocol. Each (0.41 M formaldehyde, 1X MOPS-EDTA-sodium acetate). insect was weighed to the nearest milligram after each At the end of each gel run, the gel was photographed to respirometry session. Oxygen consumption and carbon compare rRNA intensity. RNA was subsequently trans- dioxide production levels were calculated for each insect ferred overnight to a positively charged nylon membrane based on their weights and enclosure time and expressed as (Roche, Indianapolis) with downward capillary action ml/g/h. using a 20X SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) transfer buffer (Schleicher and Schuell, Keene, NH, 2.3. Survival rate, emergence, and potential fecundity of USA). The blots were air-dried, UV cross-linked at larvae subjected to long-term cold storage 12,000 mJ/cm2, and stored at 20 1C. Blots were prehy- bridized and hybridized using the DIG Easy Hybridization Data from a study conducted between 1973 and 1988 is BufferTM (Roche, Indianapolis), and screened with probes being introduced herein to provide a more complete at a concentration of 20 ng/ml of hybridization buffer. The characterization of the physiological status of these insects digoxigenin-labeled probes were detected using the DIG when subjected to long-term cold storage, and to explore High Prime DNA Labeling and Detection Start Kit II the biological potentiality of T. myopaeformis larvae to (Roche, Indianapolis). survive and reproduce after being subjected to several years of cold (671 1C) storage. Larvae used in this study were 2.5. Statistical analysis collected from sugarbeet fields during the months of July, August, and September, and were subjected to the same Respiration and days-to-pupation data were analyzed by handling procedures as described above. The larvae using one-way analysis of variance (ANOVA) (SAS intended for storage periods of 1, 2, 3, or 4 years were Institute, 2006), and mean comparisons were carried out divided into 3–34 batches, based on locality of collection. with Tukey’s multirange test. Results were expressed as ARTICLE IN PRESS 694 A. Chirumamilla et al. / Journal of Insect Physiology 54 (2008) 691–699 mean and mean+S.E.M. (standard error of mean), 3.2. Differences between laboratory-stored and field- and considered significantly different at Po0.05. Log collected diapausing larvae transformation was performed on the respirometry data from different life stages of T. myopaeformis to normalize 3.2.1. Respiration at 5 1C the distribution of data and to reduce the variation. At 5 1C, a trend towards decreased respiration was observed between field-collected November and December diapausing larvae. A similar trend was also observed from 3. Results 1 to 5 years in cold-stored larvae (Fig. 2). Oxygen use and carbon dioxide production levels indicated a significant 3.1. Respiration during developmental transition reduction (O2 from 0.04 to 0.016 ml/g/h; CO2 from 0.032 to 0.013 ml/g/h) in respiration rate from 1 to 2 years of At 20 1C, a trend of declining respiration was observed as storage; however, no significant decrease (O2 from 0.016 to the larvae entered into diapause and a significant increase 0.01 ml/g/h; CO2 from 0.013 to 0.006 ml/g/h) in respiration was seen during the adult stage (Fig. 1). Early second was observed between larvae held for 2 and 5 years of cold instars had the highest rate of respiration, making it the storage. In comparison, the oxygen consumption levels of most metabolically active developmental stage, while the 1-year stored larvae were similar to those of diapausing third-instar larvae collected in December had the lowest (November and December) larvae, but carbon dioxide larval respiration rate, indicating that they were in a deep production levels of 1-year stored larvae were only similar state of diapause. The phase of diapause initiation in to November diapausing larvae. Respiration rates of 2- and August-collected third instars was marked by a significant 5-year stored larvae resembled those of December diapaus- decrease in the oxygen consumption rate from that of late ing larvae with respect to both oxygen and carbon dioxide second instars. The transition was signaled by a 45–50% levels. Oxygen consumption exceeded carbon dioxide decrease in the total respiration rate of August-collected production in both groups of insects. third instars (O2 ¼ 0.287 ml/g/h; CO2 ¼ 0.289 ml/g/h) from that of late second instars (O2 ¼ 0.578 ml/g/h; 3.2.2. Respiration at 20 1C CO2 ¼ 0.52 ml/g/h). Respiration rates of diapausing Increasing the ambient temperature by 151 resulted in a third-instar larvae remained fairly consistent with the sharp increase in carbon dioxide production that ranged progression of diapause from August to November and from eight-fold (i.e., 0.037–0.31 ml/g/h) in November- then decreased significantly in December. The difference collected diapausing larvae to 14-fold (i.e., 0.01–0.15 ml/ between respiration rates of diapausing (December third g/h) in December-collected diapausing larvae. A 6–10-fold instars) and post-diapausing larvae was insignificant rise in oxygen consumption was observed in both diapaus- (O2 ¼ 0.133–0.183 ml/g/h; CO2 ¼ 0.126–0.186 ml/g/h). Pu- ing and stored larvae (Figs. 2 and 3). Respiration patterns pae had the lowest (O2 ¼ 0.104 ml/g/h; CO2 ¼ 0.103 ml/g/ at 20 1C clearly distinguished two groups: (1) 1-year stored h) respiration rate among all stages, and respiration was and November diapausing larvae with high respiration; significantly greater in adults. and (2) 2- and 5-year stored and December diapausing

Fig. 1. Mean oxygen consumption and carbon dioxide production of different stages of T. myopaeformis at 20 1C: E-2nd, early second instar; L- 2nd, late second instar; 3rd-Aug, third instar collected in August; 3rd-Sept, third instar collected in September; 3rd-Oct, third instar collected in Fig. 2. Differences in mean oxygen consumption and carbon dioxide October; D-1 Nov, diapausing third instar collected in November; D-2 production (mean+S.E.M.) at 5 1C between diapausing T. myopaeformis Dec, diapausing third instar collected in December; PD, post-diapause larvae of November (D-1 Nov), diapausing larvae of December (D-2 Dec), third instar in May; Pup, pupae in May; AF, adult females in June; AM, and larvae stored in the laboratory for 1, 2, or 5 years. Bars sharing a letter adult males in June. All stages of insect were collected in the year 2004. are not signiﬁcantly different (Tukey’s test). One-way ANOVA Bars sharing a letter are not signiﬁcantly different (Tukey’s test). One-way (Po0.0001). N ¼ 5 (D-2 Dec and 5 years); N ¼ 6 (D-1 Nov and 2 years); ANOVA (Po0.0001). N ¼ 6. Data ln-transformed. N ¼ 7 (1 year). Year of collection in parentheses. ARTICLE IN PRESS A. Chirumamilla et al. / Journal of Insect Physiology 54 (2008) 691–699 695 larvae with low respiration. The higher temperature tended (i.e., ranging from 74% to 84% in 1-, 2-, and 3-year stored to increase carbon dioxide production rates of both larvae), and only dropped to 62% after 4 years of storage diapausing and stored larvae to similar concentrations as (Table 1). When larvae were taken out of storage and those of oxygen consumption rates. maintained at 25 1C, 75% pupation occurred within 11–14 days in 1- and 2-year stored larvae while it took place 3.3. Survival rate, emergence, and potential fecundity of within 8 days in 3-year stored larvae (Fig. 4). It took an larvae following long-term storage additional week for 1- and 2-year stored larvae to achieve 90% pupation, and only two additional days for the 3-year 3.3.1. First sampling stored larvae to reach 90%. In order to complete the Long-term storage had little effect on pupation. The remaining 10% of the total pupation, it took an average of percentage of larvae that pupated was surprisingly high 4–5 weeks for the 1- and 2-year stored larvae, while it occurred within 2 weeks for the 3-year stored larvae. An overall summation indicates that the average number of

Fig. 3. Differences in oxygen consumption and carbon dioxide production (mean+S.E.M.) at 20 1C between diapausing T. myopaeformis larvae of Fig. 4. Number of days (mean+S.E.M.) taken for pupation (75%, 90%, November (D-1 Nov), diapausing larvae of December (D-2 Dec) and and 100%) in T. myopaeformis larvae stored in the laboratory for 1, 2, or 3 larvae stored in the laboratory for 1, 2, or 5 years. Bars sharing a letter are years. Bars with same letters are not signiﬁcantly different (Tukey’s test). not signiﬁcantly different (Tukey’s test). One-way ANOVA (Po0.0001). One-way ANOVA (Po0.0001). Number of batches with size ranging from N ¼ 6 (D-1 Nov, D-2 Dec, 2 and 5 years); N ¼ 7 (1 year). 49 to 1215 larvae per batch in parentheses.

Table 1 Survival rate, emergence, and potential fecundity of T. myopaeformis larvae following prolonged long-term storage s.e.m

s.e.m (n = 2) (n = 6) ARTICLE IN PRESS 696 A. Chirumamilla et al. / Journal of Insect Physiology 54 (2008) 691–699 days taken for the 3-year-old larvae to pupate was Expression of Fbp2 was detected in diapausing larvae, but significantly lower (24 days) than 1- and 2-year (55 and not in 1-, 2-, or 5-year stored larvae (Fig. 5a). 51 days) stored larvae (Fig. 4). The percentage of larvae that did not pupate (NP) and remained alive after 3 months 3.4.1. Effect of temperature on regulation of Fbp2 of exposure to higher temperatures and moisture condi- To determine the effect of temperature on expression of tions ranged from 11% to 17% in 1-, 2-, and 3-year stored Fbp2 in stored larvae, 1-year cold-stored (671 1C) larvae larvae, while only 5% of 4-year stored larvae failed to were exposed to 20 1C for 0, 24, 48, or 72 h. Compared to pupate (Table 1). The percentage of adult emergence diapausing control larvae, very low levels of Fbp2 declined gradually from 1 to 4 years of storage with male expression were detected at 0 h and no signal was observed survival being greater than that of females. Although the at 24, 48, or 72 h (Fig. 5b). rate of adult emergence declined with increasing time in storage, fecundity and percent hatch remained high for 4. Discussion insects held in cold storage for up to 4 years (Table 1). Diapause is a dynamic event consisting of several 3.3.2. Second sampling successive phases rather than a single static state (Tauber Data for the second sampling was available only for et al., 1986; Danks, 1987; Hodek, 1996; Denlinger, 2002). batches originating from 1- and 2-year stored larvae Entry into diapause by insects in temperate climates is (Table 1), with fewer batches and very few larvae per characterized by metabolic suppression, whereas termina- batch. In the second sampling, pupation rates of the 1- and tion of diapause involves an elevation in metabolic activity 2-year (NP) stored larvae were 67% and 58%, respectively, (Tauber et al., 1986; Danks, 1987; Kemp et al., 2004). In and of those pupated, 51% and 75% emerged as adults. our study, diapause initiation was represented by a A substantial drop in fecundity was observed in the second precipitous fall in the respiration rate of non-feeding sampling of 1- and 2-year (NP) stored larvae compared to third-instar larvae collected in August from that of late the first sampling. Interestingly, 19–24% of the larvae second instars. The trend of respiration levels being remained alive but did not pupate after the second consistently lower from August to November indicates a sampling. progression into diapause. This supports the previous reports of sugarbeet root maggot beginning diapause 3.4. Differential gene expression in August and remaining in diapause for 6 months (Callenbach et al., 1957; Harper, 1962; Klostermeyer, Differential display revealed approximately 30 gene 1973). Surprisingly, the phase of diapause termination fragments showing differential expression in diapausing was difficult to mark because there was no significant and laboratory-stored larvae. However, northern blot increase in respiration rate of post-diapause larvae from analysis confirmed differential expression of only a single that of diapausing larvae in December. This could be due gene, D20A2b. A BlastX search of the GenBank sequence to larvae passing the actual phase of diapause termination repository found Drosophila spp. fat body protein 2 gene and approaching the phase of metamorphosing into pupae, (Fbp2) as the best match in sequence similarity to D20A2b. which is supported by the fact that most of the larvae in our collection pupated immediately after the recordings. Lowered metabolic activity during metamorphosis is common in endopterygote insects. Lowered oxygen consumption during larval-pupal ecdysis has been reported in wax moth Galleria mellonella (L.) (Sehnal and Slama, 1966). Oxygen consumption during the period of metamorphosis from larva to adult through a pupal stage was shown to follow a U-shaped curve in non-diapausing Sarcophaga argyrostoma (Robineau–Desviody) at 25 1C (Denlinger et al., 1972). Reduced metabolic activity could also account for the low levels of respiration observed in sugarbeet root maggot pupae in the present study. Significant respiration increases were only observed in adults (Fig. 1). Respirometry, usually carried out measuring only oxygen consumption, has been used in the past as a reliable Fig. 5. Northern Blot analysis of (a) Fbp2 expression in field-collected tool for distinguishing diapausing from non-diapausing diapausing T. myopaeformis larvae collected in November 2004 (D) and insects because insect respiration rates are low during larvae stored in the laboratory for 1 (2003), 2 (2002), or 5 (1999) years; (b) effect of temperature on Fbp2 expression in 1-year stored larvae exposed diapause and, with some exceptions, are relatively tem- to 24 1C for 0, 24, 48, and 72 h. Ethidium bromide stained ribosomal RNA perature-independent. At 5 and 20 1C, the respiration rates is shown below its respective northern blot. of laboratory-stored larvae in this study resembled those of ARTICLE IN PRESS A. Chirumamilla et al. / Journal of Insect Physiology 54 (2008) 691–699 697

field-collected diapausing larvae. Two groups were ob- The post-storage performance data of stored larvae served: (1) larvae stored for 1 year resembled the (Fig. 4) also offers reasonable support to the explanation November-collected diapausing larvae; and (2) larvae that a majority of the long-term stored larvae are not in stored for 2 and 5 years had similar respiration levels to diapause. In this study, 90% of the larvae pupated within those of diapausing larvae collected from the field in 20 days of exposure to higher temperature and relative December (Figs. 2 and 3). This indicates that long-term humidity. The speed at which resumption of development cold-stored larvae are maintaining a low metabolic status occurred in stored larvae supports the contention that that is comparable to that of diapausing larvae. A high larvae have completed the refractory period of diapause level of respiration could be detrimental to the survival and and are maintaining a state of post-diapause quiescence in development of stored larvae through extended periods at which development can be initiated with a sufficient rise in 5 1C, especially since energy sources are limited. Moreover, temperature (Lees, 1956; Tauber et al., 1986; Danks, 1987; the metabolic similarity of stored larvae to diapausing Hodek, 1996, 2002). Respiration levels and gene expression larvae can be seen in their response to an increase in pattern also indicate that the majority of long-term stored temperature. A 151 rise in temperature elevated the overall larvae are likely in a state of post-diapause quiescence. respiration rate in both groups (Figs. 2 and 3). Increases in Post-diapausing insects in quiescence are known to have respiration with increasing temperatures have been re- the advantage of retaining all the benefits of diapause ported in diapausing pupae of Pieris brassicae (L.), with (e.g., cold-hardiness, suppressed metabolism) without respiration increasing exponentially between 5 and 20 1C having to maintain diapause (Tauber et al., 1986). (Fourche, 1977). Similarly, a 1.5-fold increase in oxygen Despite the fact that there was a high level of pupation consumption was observed in diapausing larvae of the (62–84%) in all groups of stored larvae following exposure burnet moth, Zygaena trifolii Esper, following a tempera- to higher temperatures, a fraction (5–17%) of them ture increase from 5 to 20 1C(Wipking et al., 1995). In the exhibited a dissimilar response by remaining as dormant, current study, increasing the temperature led to a 6–10-fold non-pupated larvae (Table 1). The above results clearly increase in oxygen consumption. Interestingly, carbon show that two different groups of insects were represented dioxide production rose by 8–14-fold (Figs. 2 and 3). within the stored larvae surveyed. The pupated individuals, Recent advances in gene expression studies emphasize which comprised the majority, represented post-diapause that diapause can indeed be characterized by upregulation quiescent larvae, whereas the minor group of larvae that of certain genes, in addition to a widespread gene did not pupate remained in prolonged diapause. Although shutdown (Denlinger, 2002). Differential display compar- there are no previous reports on the incidence of prolonged ing diapausing T. myopaeformis larvae with those main- diapause in T. myopaeformis, data from the second tained in cold storage for 1, 2, and 5 years clearly sampling in our study (Table 1) clearly support the demonstrated that stored larvae differ from diapausing occurrence of this phenomenon in this species. Among larvae at the molecular level, with a transcript similar to the non-pupated 1- and 2-year stored larvae subjected to Fbp2 being highly upregulated in diapausing larvae and not second sampling, 67% and 58%, respectively, pupated in being expressed at detectable levels in long-term stored the following year (second sampling), while 19% and 24%, larvae (Fig. 5a), except for traces of expression in some respectively, remained in a dormant state. Similar patterns samples of 1-year stored larvae. In Drosophila, Fbp2 is an of within-population variation in overwintering duration ancient duplication of the alcohol dehydrogenase gene have been seen in Colorado potato beetles L. decemlineata (Adh), which encodes a protein that differs substantially (Tauber and Tauber, 2002), the yucca moth, Prodoxus from ADH in its methionine content. FBP2 is functionally y-inversus Riley (Powell, 1989, 2001), and C. elephas (Soula homologous to ADH, but differs in amino acid content by and Menu, 2005), where some individuals within a being high (20%) in methionine (Meghlaoui and Veuille, population maintained diapause for extended periods. 1997). Association of Adh with dormancy was established The adaptive value of these long cycles has been suggested in nematodes where the Adh (dod-11) gene, was found as contributing to synchronized mass emergences (Powell, upregulated in dauer larvae of Caenorhabditis elegans 1989, 2001) and as a ‘‘bet-hedging’’ strategy to decrease the (Murphy et al., 2003). The fact that expression levels of a risk of population decimation by unpredictable cata- transcript for Fbp2 (D20A2b) were high in diapausing strophic events (Menu et al., 2000). larvae and low in only some stored larvae (1 year–0 h) In conclusion, the extended survival of T. myopaeformis suggests that a majority of the stored larvae are not in in long-term cold storage is facilitated by two mechanisms, diapause. In addition, the absence of Fbp2 expression diapause and post-diapause quiescence. We arrive at this upon exposure to a higher temperature in 1-year stored conclusion because all groups of stored T. myopaeformis larvae (Fig. 5b) suggests that Fbp2 is tightly diapause- included mixed populations of prolonged diapause and regulated, but not affected by environmental factors post-diapause quiescent larvae, with the majority being in such as temperature. This contrasts with the expression the quiescent state. 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