Non-diapausing Females Research Articles

Consuming royal jelly alters several phenotypes associated with overwintering dormancy in mosquitoes.

Females of the Northern house mosquito, Culex pipiens, enter an overwintering dormancy, or diapause, in response to short day lengths and low environmental temperatures that is characterized by small egg follicles and high starvation resistance. During diapause, Culex pipiens Major Royal Jelly Protein 1 ortholog (CpMRJP1) is upregulated in females of Cx. pipiens. This protein is highly abundant in royal jelly, a substance produced by honey bees (Apis mellifera), that is fed to future queens throughout larval development and induces the queen phenotype (e.g., high reproductive activity and longer lifespan). However, the role of CpMRJP1 in Cx. pipiens is unknown. We first conducted a phylogenetic analysis to determine how the sequence of CpMRJP1 compares with other species. We then investigated how supplementing the diets of both diapausing and nondiapausing females of Cx. pipiens with royal jelly affects egg follicle length, fat content, protein content, starvation resistance, and metabolic profile. We found that feeding royal jelly to females reared in long-day, diapause-averting conditions significantly reduced the egg follicle lengths and switched their metabolic profiles to be similar to diapausing females. In contrast, feeding royal jelly to females reared in short-day, diapause-inducing conditions significantly reduced lifespan and switched their metabolic profile to be similar nondiapausing mosquitoes. Moreover, RNAi directed against CpMRJPI significantly increased egg follicle length of short-day reared females, suggesting that these females averted diapause. Taken together, our data show that consuming royal jelly reverses several key seasonal phenotypes of Cx. pipiens and that these responses are likely mediated in part by CpMRJP1.

The potential for using aerated steam to eradicate strawberry mite and two-spotted spider mite on strawberry transplants.

In this study, we investigated if a steam treatment program used to produce disease-free strawberry transplants has the potential to also eliminate strawberry mite (Phytonemus pallidus) and two-spotted spider mite (Tetranychus urticae). Crowns of strawberry plants collected in a commercial field, containing young, folded leaves with all life stages of P. pallidus, and strawberry leaf discs on water agar with T. urticae with non-diapausing adult females and eggs from a laboratory rearing, were exposed to warm aerated steam in a steam cabinet in a series of four experimental runs over 2years. The steam treatments constituted of a 1-h pre-treatment with 37°C steam followed by a 1-h recovery period at 21-25°C, and then a main steam treatment at 44°C for either 2, 4 (both P. pallidus and T. urticae) or 6h (the more heat tolerant T. urticae only). After steaming, the plant material with P. pallidus or T. urticae were incubated at 21-25°C until survival was assessed after 1-6days, depending on the mite species and life-stage. Non-steamed plant material with mites was used as controls. The 4-h treatment killed all P. pallidus eggs, larvae and adults, and the 2-h treatment killed all individuals in all three stages except for one egg in one of the runs. There were no or minor effects of the steam treatments on T. urticae adult and egg survival. Based on these results, the tested steam treatments may be used to eliminate the strawberry mite but not the two-spotted spider mite from strawberry planting material.

Preimaginal caste-related bias in the paper wasp Polistes jokahamae is limited to the first brood

Whether the caste fate of social insects is determined before or after emergence is a key question for understanding the evolution of eusociality. Paper wasps are a suitable model for answering this question because there are no critical morphological differences between queens and workers in paper wasps, and these animals appear to represent an early stage of eusociality. We explored the above question by determining the effects of photoperiod during the adult stage on caste-fate determination in the paper wasp Polistes jokahamae. We collected colonies at different stages in the field and exposed emerging adults individually to long or short days. Under these isolated conditions, gyne-destined (diapausing) females were expected to exhibit large lipid stores without mature eggs, while the reverse was expected to be true for worker-destined (nondiapausing) females. The proportion of wasps with mature eggs was higher under long days in the second and subsequent broods, but not in the first brood. Lipid stores were larger among large females and under short days, and smaller for the first brood. These findings together suggest that the first brood emerges with a strong preimaginal bias toward workers (nondiapausing form), whereas the other broods emerge with no bias or an easily reversible bias. However, it is difficult to conclude whether the bias came from body size or the season of emergence. We discuss the possibility that the ancestor of paper wasps had workers with and without preimaginal bias toward becoming workers at emergence.

Biological attributes of diapausing and non-diapausing Doryctobracon areolatus (Hymenoptera, Braconidae), a parasitoid of Anastrepha spp. (Diptera, Tephritidae) fruit flies

Doryctobracon areolatus(Szépligeti), a solitary endoparasitoid native to the Neotropics, attacks eggs and early instar larvae ofAnastrephafruit flies, and can enter diapause under tropical and subtropical conditions. We aimed to test if biological attributes, such as size, flight ability, starvation resistance, longevity and fecundity of diapausing individuals differ from those of non-diapausing ones. Parasitoids were obtained from a laboratory colony reared onAnastrepha ludens(Loew) larvae. Parasitized host puparia were sorted in two cohorts according to their diapause condition. Developmental time from egg to adult ranged from 18 to 31 days in non-diapausing parasitoids, and 70 to 278 days for diapausing individuals. Pupal weight and adult measurements were higher in non-diapausing than in diapausing parasitoids. There were no differences in adult longevity, starvation resistance, and emergence between diapausing and non-diapausing wasps. Flight ability and fecundity rates were greater in the non-diapausing than in the diapause cohort. The proportion of female offspring was greater in the non-diapausing cohort (42.5%), whereas in the diapausing cohort the male offspring proportion was greater (62.4%). Both cohorts produced diapause offspring, but the non-diapausing cohort produced more (26.6%) than the diapausing one (9.1%). Maternal age had a significant effect on the proportion of diapause offspring: in 26 to 34 days old non-diapausing females, 78.9% of their offspring entered into diapause. These results confirmed that diapause affects the biological attributes ofD. areolatus. The observed differences contribute to better understand the diapause influence on the colonization and rearing process of this species and its use as biocontrol agent.

Evidence that microRNAs are part of the molecular toolkit regulating adult reproductive diapause in the mosquito, Culex pipiens.

For many insects, diapause is the primary mechanism for surviving unfavorable seasons. Some aspects of diapause regulation are well known, but we still lack a mechanistic understanding of molecular mechanisms that control the diapause pathway. Accumulating evidence suggests microRNAs regulate diapause in evolutionarily diverse insect species including flesh flies and moths, and, it is likely that microRNAs regulate multiple characteristics of diapause, including arrested egg follicle development and fat hypertrophy, in females of the Northern house mosquito, Culex pipiens. To investigate microRNA regulation of diapause in this species, we measured changes in egg follicle development and total lipid content over 22 days following adult emergence. We also evaluated changes in the abundance of candidate microRNAs associated with these physical changes during the same time frame. We found egg follicle size and lipid content were nearly the same in diapausing and nondiapausing females on the day of adult emergence, and then diverged over time such that by day 22 diapausing females had significantly smaller egg follicles and higher total lipids than their nondiapausing counterparts. Several microRNAs associated with lipid metabolism in insects, including miR-14-3p, miR-277-3p, and miR-305-5p, were underexpressed in diapausing females compared to nondiapausing females on the day of adult emergence, which suggests microRNA regulation occurs ahead of observed changes in these two features of the diapause phenotype. We also found miR-309-3p, miR-375-3p which stimulate ovarian development in other mosquito species, were underexpressed in diapausing females of Cx. pipiens at times after diapause is fully established and may be responsible for the arrest in ovarian development in this species. Taken together, our results demonstrate that changes in the abundance of some microRNAs is associated with phenotypic changes in diapause Cx. pipiens and suggests this epigenetic mechanism is part of the molecular toolkit regulating diapause.

Life-history changes in the cold tolerance of the two-spot spider mite Tetranychus urticae: applications in pest control and establishment risk assessment.

Lethal time50 (LTime50) and lethal temp (LTemp50) are commonly used laboratory indices of arthropod cold tolerance, with the former often being employed to predict winter survival in the field. In the present study, we compare the cold tolerance of different life‐history stages (nondiapausing and diapausing females, as well as males and juveniles) of a major agricultural pest: the two‐spot spider mite Tetranychus urticae Koch (Acarina: Tetranychidae). Diapausing females from European populations of this species are shown to be freeze avoiding, supercooling to −23.6 ± 0.37 °C and with an LTemp50 of −23.2 °C. However, nondiapausing females [supercooling point (SCP) –19.1 ± 0.49 °C, LTemp50 –14.32 °C], males (SCP –21.27 ± 0.52 °C, LTemp50 –16 °C) and juveniles (SCP –25.34 ± 0.29 °C, LTemp50 –18.3 °C) are subclassified as strongly chill tolerant juveniles. LTime50 is 148.3 days for non‐acclimated diapausing females, whereas nondiapausing females, males and juveniles reach 50% mortality by 21.7 days. When individuals are acclimated at 10 °C for a period of 7 days, no effect is found. Cold tolerance is suggested to be a major contributor to the successful spread of T. urticae across temperate countries, although it is dependent on a diapause trait, suggesting a potential target for control. Winter field trial data from diapausing females indicate that LTime50 is a reliable indicator of winter survival even within diapause, supporting the use of these indices as a valuable component within environmental niche models for the prediction of future pest invasions.

Non-Destructive Detection of Diapause in the Western Tarnished Plant Bug, Lygus hesperus Knight (Hemiptera: Miridae)

The western tarnished plant bug, Lygus hesperus Knight, overwinters in an adult diapause. However, the ecological implications of diapause to L. hesperus population survival are poorly understood. Enhanced understanding of L. hesperus overwintering ecology might reveal opportunities to develop ecologically-based management tactics. One factor limiting efforts to understand the dynamics of overwintering is lack of a non-destructive method to distinguish diapause. We evaluated a suite of external characters for utility in distinguishing diapausing from non-diapausing L. hesperus. Abdomen coloration of female L. hesperus was a highly reliable indicator of diapause status. During these studies, a single female (0.6%) was misclassified using the abdomen color criterion. For male L. hesperus, corrected abdomen length (abdomen length / head capsule width) was used to predict diapause status with ≈84% accuracy. Both criteria provided improved accuracy compared with earlier reports, in part because their application was limited to adults of a specific age (10 d) and reared at a specific temperature (26.7°C). Application of these criteria to individuals that were subsequently starved allowed us to unambiguously distinguish the survival functions of diapausing and non-diapausing males and females. Although neither criterion (abdomen color for females, corrected abdomen length for males) was error-free, both offer sufficient accuracy to justify their use in studies of the ecology, physiology, or molecular biology of L. hesperus diapause. These criteria provide the ability to non-destructively distinguish the diapause status of adult L. hesperus with reasonable accuracy, and should enhance efforts to better understand diapause and overwintering ecology in this important pest species.

The diapause program impacts renal excretion and molecular expression of aquaporins in the northern house mosquito, Culex pipiens

Adult females of the mosquito Culex pipiens entering diapause increase sugar water ingestion and reduce evaporative water loss, but how these attributes of the diapause program impact activity of the renal excretory system remains unknown. Here we compared the renal excretory capacity of diapausing and non-diapausing females, as well as the molecular expression of aquaporin (AQP) genes that encode channels involved in transporting water and/or small metabolites. Baseline urine excretion rates in diapausing mosquitoes were higher than in those of their non-diapausing counterparts, possibly a consequence of the intense sugar feeding associated with diapause. But, diapausing mosquitoes exhibited a much lower capacity for diuresis than non-diapausing mosquitoes. The suppressed diuretic capacity likely reflects reduced investment in the energetically-expensive post-prandial diuresis, an event not observed in diapausing mosquitoes. The mRNA expression levels of two genes encoding AQPs, Eglp1 and Aqp12L, in diapausing mosquitoes were down-regulated (on day 14) and up-regulated (on both days 3 and 14), respectively, in whole body samples. These changes were not evident in the excretory system (i.e., Malpighian tubules and hindgut), which showed no differential expression of AQPs as a function of diapause. Several AQP mRNAs were, however, differentially expressed in the midgut, ovaries, and abdominal body wall of diapausing mosquitoes, suggesting that AQPs in these tissues may be playing important non-excretory roles that are unique to diapause physiology.

Transcriptional Differences between Diapausing and Non-Diapausing D. montana Females Reared under the Same Photoperiod and Temperature.

BackgroundA wide range of insects living at higher latitudes enter diapause at the end of the warm season, which increases their chances of survival through harsh winter conditions. In this study we used RNA sequencing to identify genes involved in adult reproductive diapause in a northern fly species, Drosophila montana. Both diapausing and non-diapausing flies were reared under a critical day length and temperature, where about half of the emerging females enter diapause enabling us to eliminate the effects of varying environmental conditions on gene expression patterns of the two types of female flies.ResultsRNA sequencing revealed large differences between gene expression patterns of diapausing and non-diapausing females, especially in genes involved with metabolism, fatty acid biosynthesis, and metal and nucleotide binding. Differently expressed genes included several gene groups, including myosin, actin and cytochromeP450 genes, which have been previously associated with diapause. This study also identified new candidate genes, including some involved in cuticular hydrocarbon synthesis or regulation (desat1 and desat2), and acyl-CoA Δ11-desaturase activity (CG9747), and few odorant-binding protein genes (e.g. Obp44A). Also, several transposable elements (TEs) showed differential expression between the two female groups motivating future research on their roles in diapause.ConclusionsOur results demonstrate that the adult reproductive diapause in D. montana involves changes in the expression level of a variety of genes involved in key processes (e.g. metabolism and fatty acid biosynthesis) which help diapausing females to cope with overwintering. This is consistent with the view that diapause is a complex adaptive phenotype where not only sexual maturation is arrested, but also changes in adult physiology are required in order to survive over the winter.

Females of Riptortus pedestris (Hemiptera: Alydidae) in Reproductive Diapause are More Responsive to Synthetic Aggregation Pheromone.

Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) enters reproductive diapause under short-day length (< 13.5 h) conditions. As an apparent increase of field populations of R. pedestris has been observed during the fall when aggregation pheromone traps are placed in soybean fields, we tested the hypothesis that R. pedestris becomes more responsive to the aggregation pheromone during fall as it enters its reproductive diapause. The response of R. pedestris females-either ones collected seasonally from fields or reared in the laboratory under short-day length (10:14 [L:D] h) conditions-to the aggregation pheromone was examined using a Y-tube olfactometer. Riptortus pedestris collected in fall showed a higher response to the aggregation pheromone (74-80%) than those collected in July (40%). Females in which diapause was induced in the laboratory also showed a higher response to the pheromone than those not in diapause. In a wind tunnel assay, female bugs in laboratory-induced diapause also responded positively to the aggregation pheromone. In addition, we assessed the reproduction of females to verify their diapausing status. Diapausing females never mated with either short- or long-day-reared males, nor did they reproduce, but non-diapausing females mated with short-day-reared males at a rate of 13%. Females collected from fields during fall never mated. In a soybean field evaluation, the number of adult female R. pedestris was higher in the presence of an aggregation pheromone trap than in the absence of one. This is the first confirmation of higher response to the aggregation pheromone of both field-collected and laboratory-reared R. pedestris in reproductive diapause.

Circadian clock of Drosophila montana is adapted to high variation in summer day lengths and temperatures prevailing at high latitudes

Photoperiodic regulation of the circadian rhythms in insect locomotor activity has been studied in several species, but seasonal entrainment of these rhythms is still poorly understood. We have traced the entrainment of activity rhythm of northern Drosophila montana flies in a climate chamber mimicking the photoperiods and day and night temperatures that the flies encounter in northern Finland during the summer. The experiment was started by transferring freshly emerged females into the chamber in early and late summer conditions to obtain both non-diapausing and diapausing females for the studies. The locomotor activity of the females and daily changes in the expression levels of two core circadian clock genes, timeless and period, in their heads were measured at different times of summer. The study revealed several features in fly rhythmicity that are likely to help the flies to cope with high variation in the day length and temperature typical to northern summers. First, both the non-diapausing and the diapausing females showed evening activity, which decreased towards the short day length as observed in the autumn in nature. Second, timeless and period genes showed concordant daily oscillations and seasonal shifts in their expression level in both types of females. Contrary to Drosophila melanogaster, oscillation profiles of these genes were similar to each other in all conditions, including the extremely long days in early summer and the cool temperatures in late summer, and their peak expression levels were not locked to lights-off transition in any photoperiod. Third, the diapausing females were less active than the non-diapausing ones, in spite of their younger age. Overall, the study showed that D. montana clock functions well under long day conditions, and that both the photoperiod and the daily temperature cycles are important zeitgebers for seasonal changes in the circadian rhythm of this species.

Drosophila americana Diapausing Females Show Features Typical of Young Flies.

Diapause is a period of arrested development which is controlled physiologically, preprogrammed environmentally and characterized by metabolic depression that can occur during any stage of insect development. Nevertheless, in the genus Drosophila, diapause is almost always associated with the cessation of ovarian development and reproductive activity in adult females. In this work, we show that, in D. americana (a temperate species of the virilis group), diapause is a genetically determined delay in ovarian development that is triggered by temperature and/or photoperiod. Moreover, we show that in this species diapause incidence increases with latitude, ranging from 13% in the southernmost to 91% in the northernmost range of the distribution. When exposed to diapause inducing conditions, both diapausing and non-diapausing females show a 10% increase in lifespan, that is further increased by 18.6% in diapausing females, although senescence is far from being negligible. ActinD1 expression levels suggest that diapausing females are biologically much younger than their chronological age, and that the fly as a whole, rather than the ovarian development alone, which is phenotypically more evident, is delayed by diapause. Therefore, diapause candidate genes that show expression levels that are compatible with flies younger than their chronological age may not necessarily play a role in reproductive diapause and in adaptation to seasonally varying environmental conditions.

Seasonal gene expression kinetics between diapause phases in Drosophila virilis group species and overwintering differences between diapausing and non-diapausing females.

Most northern insect species experience a period of developmental arrest, diapause, which enables them to survive over the winter and postpone reproduction until favorable conditions. We studied the timing of reproductive diapause and its long-term effects on the cold tolerance of Drosophila montana, D. littoralis and D. ezoana females in seasonally varying environmental conditions. At the same time we traced expression levels of 219 genes in D. montana using a custom-made microarray. We show that the seasonal switch to reproductive diapause occurs over a short time period, and that overwintering in reproductive diapause has long-lasting effects on cold tolerance. Some genes, such as Hsc70, Jon25Bi and period, were upregulated throughout the diapause, while others, including regucalcin, couch potato and Thor, were upregulated only at its specific phases. Some of the expression patterns induced during the sensitive stage, when the females either enter diapause or not, remained induced regardless of the later conditions. qPCR analyses confirmed the findings of the microarray analysis in D. montana and revealed similar gene expression changes in D. littoralis and D. ezoana. The present study helps to achieve a better understanding of the genetic regulation of diapause and of the plasticity of seasonal responses in general.

Deciphering the metabolic changes associated with diapause syndrome and cold acclimation in the two-spotted spider mite Tetranychus urticae.

Diapause is a common feature in several arthropod species that are subject to unfavorable growing seasons. The range of environmental cues that trigger the onset and termination of diapause, in addition to associated hormonal, biochemical, and molecular changes, have been studied extensively in recent years; however, such information is only available for a few insect species. Diapause and cold hardening usually occur together in overwintering arthropods, and can be characterized by recording changes to the wealth of molecules present in the tissue, hemolymph, or whole body of organisms. Recent technological advances, such as high throughput screening and quantification of metabolites via chromatographic analyses, are able to identify such molecules. In the present work, we examined the survival ability of diapausing and non-diapausing females of the two-spotted spider mite, Tetranychus urticae, in the presence (0 or 5°C) or absence of cold acclimation. Furthermore, we examined the metabolic fingerprints of these specimens via gas chromatography-mass spectrophotometry (GC-MS). Partial Least Square Discriminant Analysis (PLS-DA) of metabolites revealed that major metabolic variations were related to diapause, indicating in a clear cut-off between diapausing and non-diapausing females, regardless of acclimation state. Signs of metabolic depression were evident in diapausing females, with most amino acids and TCA cycle intermediates being significantly reduced. Out of the 40 accurately quantified metabolites, seven metabolites remained elevated or were accumulated in diapausing mites, i.e. cadaverine, gluconolactone, glucose, inositol, maltose, mannitol and sorbitol. The capacity to accumulate winter polyols during cold-acclimation was restricted to diapausing females. We conclude that the induction of increased cold hardiness in this species is associated with the diapause syndrome, rather than being a direct effect of low temperature. Our results provide novel information about biochemical events related to the cold hardening process in the two-spotted spider mite.

Abiotic and Biotic Factors Affecting Resting Spore Formation in the Mite PathogenNeozygites floridana

Neozygites floridana is an obligate mite pathogenic fungus in the Entomophthoromycota. It has been suggested that resting spores of this fungus are produced as a strategy to survive adverse conditions. In the present study, possible mechanisms involved in the regulation of resting spore formation were investigated in the hosts Tetranychus urticae and Tetranychus evansi. Abiotic and biotic factors mimicking conditions that we, based on earlier field studies, thought might induce resting spores in temperate and tropical regions were tested with isolates from Norway and Brazil. A total of 42 combinations of conditions were tested, but only one induced the formation of a high number of resting spores in only one isolate. The Brazilian isolate ESALQ1420 produced a large number of resting spores (51.5%) in T. urticae at a temperature of 11°C, photoperiod of 10L:14D, and light intensity of 42–46 (μmol m−2 s−1) on nonsenescent plants (nondiapausing females). Resting spores of the Brazilian N. floridana isolate ESALQ1421 were found at very low levels (up to 1.0%). Small percentages of T. urticae with resting spores (0–5.0%) were found for the Norwegian isolate NCRI271/04 under the conditions tested. The percentages of resting spores found for the Norwegian isolate in our laboratory studies are similar to the prevalence reported in earlier field studies.

Performance of diapausing parasitoid wasps, Habrobracon hebetor, after cold storage

The ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) is an important potential biological control agent for lepidopterous pests of stored products. We investigated the effects of long-term cold storage of diapausing and nondiapausing H. hebetor on their performance after cold storage. Mortality during storage increased with increasing storage duration, and the mortality of diapausing females was lower than that of nondiapausing females after 8, 12, and 16weeks of storage. Longevity, egg laying, number of progeny produced, and time to 50% egg laying were all reduced, as compared with the culture females when parasitoids were reared at conditions that do not induce diapause. But, for females reared at 20°C at conditions that induce diapause, all of these quality parameters did not differ from those of culture insects when the storage duration was 8weeks or less. The percentage of female F1 offspring was always lower for cold stored insects than for the culture insects. Presence of a male after cold storage did not impact any of the quality parameters measured. Thus, rearing parasitoids at 20°C and 10L:14D and then storing them for up to 8weeks at 5°C would produce parasitoids that are similar to culture parasitoids, except that the percentage of females is lower than that in the cultures (36% vs. 52%).

Juvenile hormone III suppresses forkhead of transcription factor in the fat body and reduces fat accumulation in the diapausing mosquito, Culex pipiens

Juvenile hormone (JH) controls diverse physiological and developmental events including diapause and nutrient metabolism. The focal point of endocrine regulation in adult reproductive diapause is initiated by a halt of JH synthesis. In diapausing females of the mosquito Culex pipiens, the other key molecular event is the signalling pathway from insulin to forkhead of transcription factor (FOXO). We hypothesized that a halt of JH synthesis is related to activation of FOXO, which results in increasing lipid reserves in the fat body at the onset of the diapause programme. In this study, the full-length sequence of the foxo gene in C. pipiens was characterized, and the protein abundance pattern of the foxo gene product was analyzed by immunoblotting and immunohistochemistry. FOXO was much more abundant in the fat body of diapausing females than in the fat body of nondiapausing females; much lower levels were present in other adult tissues. When we topically applied JH III to diapause-destined females, FOXO was suppressed, and fat accumulation was reduced, suggesting an interaction between JH synthesis and FOXO that is critical for expression of the diapause phenotype.

Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae, is a worldwide pest species that overwinters as diapausing females. Cold hardening is presumed to start during diapause development to ensure the successful overwintering of this species. To address this hypothesis, we compared cold tolerance between non-diapausing and diapausing females. We measured supercooling point (SCP) and survival to acute cold stress by exposing the mites at a range of sub-zero temperatures (from −4 to −28°C for 2h). The mean SCPs of non-diapausing and diapausing females were −19.6±0.5 and −24.7±0.3°C respectively, and freezing killed the mites. Diapausing females were significantly more cold tolerant than non-diapausing ones, with LT50 of −19.7 and −13.3°C, respectively. Further, we also examined the effects of cold acclimation (10d at 0 or 5°C) in non-diapausing and diapausing females. Our findings indicated that diapause decreased SCP significantly, while cold acclimation had no effect on the SCP except for non-diapausing females that were acclimated at 5°C. Acclimation at 5°C enhanced survival to acute cold stress in diapausing and non-diapausing females, with LT50 of −22.0 and −17.1°C, respectively. Altogether, our results indicate that T. urticae is a chill tolerant species, and that diapause and cold acclimation elevate cold hardiness in this species.

Photoperiodic regulation of cold tolerance and expression levels of regucalcin gene in Drosophila montana

Temperature-induced plasticity of cold tolerance has been reported in many insect species, but cold tolerance can also be affected by changes in day (or night) length. In the present study we elucidate the direct and indirect effects of photoperiod on the cold tolerance of females of two Drosophila montana strains––one which possesses a robust photoperiodic diapause and another which does not. In the diapause-strain the time needed for recovery from chill coma showed a positive correlation with day length, but diapause itself played only a minor role in photoperiodic acclimation. The strain that was not able to enter to diapause as a response to day length also lacked photoperiodic cold acclimation ability indicating that this strain has deficiencies in its photoperiodic time measurement system. In the diapause-strain, the expression level of regucalcin gene was more than two times higher in diapausing than in non-diapausing females maintained in a single photoperiod, but day length per se did not cause significant changes in expression levels of this gene in either of the strains. In the non-diapausing strain this gene showed no expression changes in any comparison. Overall, the study shows that a decrease in day length can induce cold acclimation in D. montana, while changes in regucalcin expression are linked with photoperiodic diapause.

Sequence variation in couch potato and its effects on life-history traits in a northern malt fly, Drosophila montana

Couch potato (cpo) has previously been connected to reproductive diapause in several insect species including Drosophila melanogaster, where it has been suggested to provide a link between the insulin signalling pathway and the hormonal control of diapause. In the first part of the study we sequenced nearly 3.6kb of this gene in a northern Drosophila species (Drosophila montana) with a robust photoperiodically determined diapause and found several types of polymorphisms along the sequenced area. We also found variation among five Drosophila virilis group species in the length of the 5th exon of cpo and in the site of the stop codon at the end of this exon. The second part of the study was targeted on a deletion of six amino acids located in the last section of exon 5, which in D. melanogaster, is translated only in one short transcript lacking the following exons. The studied deletion appeared to be extremely rare in the wild D. montana population where it was found, but its frequency rapidly increased during laboratory culture. qPCR analyses showed the expression level of the deletion allele to be significantly downregulated in both the diapausing and non-diapausing females compared to the wild type allele. At the phenotypic level, the deletion and the decreased expression of cpo transcript involving it did not have direct effect on the incidence of female reproductive diapause, but it was associated with a reduction in development time under diapause-inducing conditions. This suggests that while the cpo transcript containing the prolonged version of the 5th exon with a stop codon is clearly associated with fly development time, the exons with RNA domains included in other transcripts of the gene may be more directly related to diapause regulation.