BACKGROUND:homologues The Pyrrhocoris apterus (Insecta: Heteroptera) adults attain high levels of cold tolerance during their overwintering diapause. Non-diapause reproducing adults, however,mRNA lack the capacity to express a whole array of cold-tolerance adaptations and show relatively low survival when exposed to sub-zero and temperatures. We assessed the competence of non-diapause males of P. apterus for responding to heat- and cold-stresses by up-regulation of overwintering 70 kDa heat shock proteins (Hsps) and the role of Hsps during repair of heat- and cold-induced injury. PRINCIPAL FINDINGS:BACKGROUND: The fragments of P. apterus homologues of Hsp70 inducible (PaHsp70) and cognate forms (PaHsc70) were cloned and sequenced. The abundance slightly of mRNA transcripts for the inducible form (qPCR) and corresponding protein (Western blotting) were significantly up-regulated in response to high protein and low temperature stimuli. In the cognate form, mRNA was slightly up-regulated in response to both stressors but very low shock, or no up-regulation of protein was apparent after heat- or cold-stress, respectively. Injection of 695 bp-long Pahsp70 dsRNA (RNAi) caused proteins drastic suppression of the heat- and cold-stress-induced Pahsp70 mRNA response and the up-regulation of corresponding protein was practically eliminated. Our Pahsp70 RNAi predictably prevented recovery from heat shock and, in addition, negatively influenced repair of chilling injuries caused by cold stress.in Cold tolerance increased when the insects were first exposed to a mild heat shock, in order to trigger the up-regulation and of PaHsp70, and subsequently exposed to cold stress. CONCLUSION: Our results suggest that accumulation of PaHsp70 belongs to a complex of cold tolerance adaptation in the insect Pyrrhocoris apterus.

To both test the hypothesis that long-term survival of sugarbeet root maggot in storage is facilitated by larvae undergoing prolonged diapause, respiration and and gene expression patterns of field-collected diapausing larvae were compared with those of 1-, 2-, and 5-year laboratory-stored larvae. Additional with assessments were made on post-storage survival, emergence, and reproductive fitness of stored larvae. Respirometry, carried out at 5 and 20 by degrees C revealed no differences among respiration rates of initially diapausing and long-term stored larvae. A 15 degrees increase in To temperature elevated respiration in both diapausing and stored larvae, with levels of CO(2) release ranging between 8- and 14-fold higher to at 20 degrees C than at 5 degrees C. Similarly, 6-10-fold increases in O(2) consumption levels were observed at the degrees higher temperature. A transcript with sequence similarity to the fat body protein 2 (Fbp2) gene was highly expressed in diapausing Therefore, larvae, and trace levels were expressed in some samples of 1-year stored larvae. However, no expression was detected in 2-no and 5-year stored larvae. Survival and emergence studies of stored larvae revealed mixed populations of diapausing (i.e., the 5-17% of However, larvae that did not pupate) and post-diapausing (62-84% of larvae pupated) insects, with a high incidence of pupation (62%) and fat emergence (47%), even after 4 years in cold storage. Therefore, extended survival of Tetanops myopaeformis larvae in long-term cold storage at is facilitated by two mechanisms, with a majority of larvae in post-diapause quiescence and a smaller fraction in a state 1-year of prolonged diapause.

When a insects are exposed to fluctuating thermal regimes (FTRs)(i.e., cold exposure alternating with periodic short pulses to high temperature), in proteins contrast to constant low temperature (CLT), mortality due to accumulation of chill injuries is markedly reduced. To investigate the physiological high processes behind the positive impact of FTR, based on a holistic approach, two-dimensional electrophoresis (2-DE) analysis were performed with the (i.e., parasitic wasp Aphidius colemani. Parasitoid proteomes revealed 369 well-distinguishable protein spots, where the overall response to cold exposure was clearly When specific to treatments (CLT versus FTR). The reduced mortality under FTR was associated with up-regulation of several proteins playing key to roles in energy metabolism (glycolysis, TCA cycle, synthesis and conversion of ATP), protein chaperoning (Hsp70/Hsp90), and protein degradation (proteasome). Our colemani. results also support the idea that cytoskeleton components, particularly actin arrangement, could play a role in the higher survival rates idea of insects under FTR.

Reproductive (in diapause was characterized in females of Pyrrhocoris apterus using physiological parameters (diapause intensity, photoperiodic responsiveness, oxygen consumption, mass and hydration)constant and changes in relative abundance of mRNA transcripts of eight different genes coding for proteins implemented in energy metabolism, cryoprotectant mRNA biosynthesis, biological clocks, and hormonal receptors. Changes in diapause intensity served as a basis for distinguishing successive phases of diapause intensity, development, which were driven both endogenously (under constant environmental conditions) and exogenously (in response to a change in environmental conditions).Reproductive Changes in the relative levels of transcripts of genes coding for aldose reductase (AR) and sorbitol dehydrogenase (SoDH) closely matched molecular those of diapause intensity and thus appeared as promising molecular markers of diapause and its development. During the initiation phase,reductase the intensity of diapause and the levels of AR and SoDH transcripts increased and reached a maximum. During maintenance, under and a constant temperature of 20 degrees C and short-day photoperiod, the intensity of diapause and the levels of both transcripts for first decreased and, later, were maintained constant. Termination of diapause was stimulated by cold, during which the intensity of diapause increased and the levels of both transcripts further decreased. Upon resumption of direct development (oogenesis, mating and oviposition), the relative abundances of of AR and SoDH transcripts decreased to trace levels.

Partial pupae clones of Megachile rotundata HSP90, HSP70, HSC70 and actin were developed by RT-PCR. These clones were used to generate probes degrees to screen for the expression of their respective transcripts in heat-shocked pupae and in diapausing prepupae through post-diapausing pupae. Northern the blot analysis revealed transcript sizes for MrHSP90, MrHSP70, MrHSC70, and Mractin of 3.6, 2.3, 2.5, and 1.4kb, respectively. MrHSP90 and by MrHSP70 were highly upregulated in post-diapausing pupae exposed to 40 degrees C for 1h, while MrHSC70 was only slightly induced Partial by heat shock. Levels of MrHSC70 and MrHSP90 showed little change between field collected diapausing prepupae and post-diapausing pupae. In contrast, contrast, MrHSP70 was highly upregulated in diapausing prepupae and Mractin was at or below the level of detection in diapausing MrHSC70 prepupae. Transferring field reared overwintering prepupae in February to 25 degrees C for 3 days induced an expression pattern of possible MrHSP70 and Mractin more typical of post-diapausing bees, indicating the likelihood that the transition to post-diapause development had occurred prior and to February. However, measurements revealed possible cyclic respiration patterns, including low respiratory quotient (RQ) values during February and March and Transferring a transition during April to more continuous respiration with elevated RQ values.

Osmia rates lignaria is a solitary bee that overwinters as a fully-eclosed, cocooned, unfed adult. Our objective is to understand the effect a of wintering temperature on diapause maintenance and termination in this species. We measure respiration rates and weight loss in individuals We exposed to various wintering temperatures ( , 4, 7, 22 degrees C, outdoors) and durations (28, 84, 140, 196, 252 days).objective We use time to emerge and respiration response (respiration rate measured at 22 degrees C) as indicators of diapause intensity.Osmia Adults spontaneously lower their respiration rates to approximately .1ml/g*h within one month after adult eclosion, indicating obligatory diapause. Non-wintered individuals a maintain low respiration rates, but lose weight rapidly and die by mid-winter. In wintered adults, two phases can be distinguished.by First, respiration response undergoes a rapid increase and then reaches a plateau. This phase is similar in bees wintered at lower , 4 and 7 degrees C. In the second phase, respiration response undergoes an exponential increase, which is more pronounced emerge at the warmer temperatures. Composite exponential functions provide a good fit to the observed respiration patterns. Adults whose respiration response which has reached .45ml/g*h emerge promptly when exposed to 20 degrees C, indicating diapause completion. Individuals wintered for short periods do This not reach such respiration levels. When exposed to 20 degrees C these individuals lower their metabolic rate, and their emergence and time is extended. The relationship between respiration rates and emergence time follows a negative exponential function. We propose two alternative an models of diapause termination to interpret these results.

Chalkbrood four is a serious disease of alfalfa leafcutting bee Megachile rotundata (F.)(Hymenoptera: Megachilidae) larvae, causing upward of 20% infection in solution the field. The causative agent is the fungus Ascosphaera aggregata. This bee is used extensively for alfalfa seed pollination in causative the United States. Using laboratory bioassays, we previously demonstrated that fungicides can reduce chalkbrood levels in the larvae. Here, we (F.) evaluate the toxicity of four fungicides, Benlate, Captan, Orbit, and Rovral, to adult bees by using three different bioassays. In Chalkbrood the first test, fungicides were applied to bees' thoraces. In the second test, mimicking foliage residue, a piece of filter of paper soaked in fungicide was placed on the bottom of a container of bees. The third test evaluated oral toxicity test, by incorporating fungicides into a sugar-water solution that was fed to the bees. The filter paper test did not discriminate The among the fungicides well, and the oral test resulted in the greatest mortality. Toxicity to males was greater than to demonstrated females. The use of fungicides for chalkbrood control is a logical choice, but caution should be used in how they oral are applied in the presence of bees.

Chalkbrood,A. a fungal disease in bees, is caused by several species of Ascosphaera. A. aggregata is a major mortality factor in in populations of the alfalfa leafcutting bee, Megachile rotundata (F.)(Hymenoptera: Megachilidae) used in commercial alfalfa seed production. Four formulated fungicides,of Benlate 50 WP, Captan, Orbit, and Rovral 50 WP were tested in the laboratory for efficacy against hyphal growth of of A. aggregata cultures. The same fungicides, with the addition of Rovral 4 F, were tested for their effects on incidence Chalkbrood, of chalkbrood disease, and toxicity to M. rotundata larvae. Benlate, Rovral 50 WP, and Rovral 4 F reduced incidence of WP, chalkbrood with minimal mortality on larval bees. Benlate and Rovral 50 WP also reduced hyphal growth. Orbit was effective in on reducing hyphal growth, but it did not reduce incidence of chalkbrood and was toxic to bee larvae. Captan was not toxic effective in reducing hyphal growth or chalkbrood incidence, and it was toxic to bee larvae. Fungicides that reduce incidence of and chalkbrood and larval mortality in this laboratory study are candidates for further study for chalkbrood control.

During orchard orchard pollination studies in California, we observed dramatic changes in nesting and foraging behavior of Osmia lignaria Say (Hymenoptera: Megachilidae)in after sprays with tank mixtures containing fungicides. A characteristic pattern of postspray events observed includes erratic behavior and interrupted foraging events and nesting activity for several days. In an effort to determine whether fungicidal sprays were disruptive to bee foraging and of thus to pollination, we exposed O. lignaria females nesting in field cages planted with lacy scorpionweed, Phacelia tanacetifolia Benth (Hydrophyllaceae),During to selected spray mixtures normally encountered in California orchard production systems: iprodione (Rovral), propiconazole (Orbit), benomyl (Benlate), and captan (Captan acted 50 WP); the surfactant Dyne-Amic, alone and mixed with Rovral; and the tank mixture IDB (Rovral + Dyne-Amic + the mixture foliar fertilizer Bayfolan Plus). An additional cage sprayed with an equal volume of water acted as control, and a cage or sprayed with the insecticide dimethoate as a toxic standard. For each female O. lignaria, we recorded time spent inside the lignaria nest depositing pollen-nectar loads, foraging time, cell production rate, and survival. All females in the dimethoate treatment died postspray +depositing 1 d. Before death, some of these females behaved similarly to our previous orchard observations. A high proportion of females as in the IDB cage were inactive for a few hours before resuming normal foraging and nesting activity. No lethal or the behavioral effects were found for any of the other compounds or mixtures tested. Our results indicate that the fungicide applications spent that we tested are compatible with the use of O. lignaria as an orchard pollinator.

The temperature solitary bee Osmia lignaria Say continues to be developed as an orchard pollinator in the United States and southern Canada.of Female bees are active during the early spring and construct nests consisting of a linear series of unlined cells delimited linear by mud partitions. Cells are provisioned with a pollen/nectar mass on which an egg is deposited, and nests are sealed States with a mud plug. In 1997, we initiated two experiments on the development, mortality, and emergence of O. lignaria at The selected laboratory temperature regimes and outdoors. In the first experiment (published previously), we compared temperature treatments for their adequacy in prepupa maintaining healthy O. lignaria populations. In a second experiment (reported here), we determined the relationship between rearing temperatures and prepupa-adult rates development rates as well as emergence time and longevity after wintering and incubation the following spring. We observed important differences many in O. lignaria prepupa versus pupa responses to selected temperature treatments. The relationship between temperature and days to pupa was the U-shaped, with additional time to transit the prepupa-pupa interval at temperatures above and below 26 degrees C. The negative relationship and between temperature and the length of the pupa to adult interval contrasts with the U-shaped thermal response observed for prepupae.pupa Thus, with each increase in thermal heat units over the range of temperature treatments tested, we observed an additional reduction prepupa-adult in the pupa-adult interval. Individuals reared at constant 18 degrees C required 2.4 times as many days to transit the interval pupa-adult interval compared with those at constant 32 degrees C. Our results suggest that there is a need for the of development of regionally adapted, latitude-specific breeding populations of this outstanding orchard crop pollinator.

The The development of a bee species as a new crop pollinator starts with the identification of a pollination-limited crop production deficit scale and the selection of one or more candidate pollinator species. The process continues with a series of studies on the one developmental biology, pollinating efficacy, nesting behaviour, preference for different nesting substrates, and population dynamics of the candidate pollinator. Parallel studies with investigate the biology of parasites, predators and pathogens. The information gained in these studies is combined with information on the The reproductive biology of the crop to design a management system. Complete management systems should provide guidelines on rearing and releasing required methods, bee densities required for adequate pollination, nesting materials, and control against parasites, predators and pathogens. Management systems should also a provide methods to ensure a reliable pollinator supply. Pilot tests on a commercial scale are then conducted to test and illustrated eventually refine the management system. The process culminates with the delivery of a viable system to manage and sustain the and new pollinator on a commercial scale. The process is illustrated by the development of three mason bees, Osmia cornifrons (Radoszkowski),a O. lignaria Say and O. cornuta (Latreille) as orchard pollinators in Japan, the USA and Europe, respectively.

One the hundred seventy human uropathogenic Escherichia coli (UPEC) clinical isolates were compared with 35 E. coli strains isolated from feces of presented a control group to determine the presence of the set1, sen and astA genes encoding the ShET-1, ShET-2, and EAST isolates toxins, respectively. Overall, 27 (16%), 8 (8%) and UPEC isolates presented the set1B, the astA, and the sen genes,uropathogenic respectively. This is the first time the set gene has been found in UPEC clinical isolates.

OBJECTIVES:and To evaluate cervical length and gestational age as predictors of intra-amniotic inflammation in patients admitted because of preterm labor and and intact membranes. METHODS: Ninety-three pregnant women with preterm labor and intact membranes were included in our study. Cervical length was admission measured on admission by transvaginal sonography and transabdominal amniocentesis was performed within the first 48 h following admission. Positive amniotic intact fluid cultures defined intra-amniotic infection. Levels of intra-amniotic interleukin-6 (IL-6) were measured, and a receiver-operating characteristics (ROC) curve was constructed OBJECTIVES: to determine the best cut-off point of IL-6 for predicting intra-amniotic infection. This value was then used as a basis ng/mL for determining a cut-off of IL-6 for defining intra-amniotic inflammation. Considering inflammatory status, perinatal outcomes were evaluated and compared. Logistic intact regression was used to investigate associations of different explanatory variables with inflammatory status. A non-invasive approach for detection of intra-amniotic diagnostic inflammation in women admitted because of preterm labor with intact membranes was evaluated. RESULTS: Intra-amniotic infection and inflammation rates were IL-6 14% and 28%, respectively. ROC curve analysis showed that the best cut-off value for IL-6 was 13.4 ng/mL for predicting developed intra-amniotic infection, which was comparable to the cut-off of 11.3 ng/mL reported previously by other authors (which we used to predicting define inflammation). Regardless of the intra-amniotic microbial status, perinatal outcomes in women who developed intra-amniotic inflammation were worse than in labor those who did not. Cervical length < 15 mm and gestational age at admission < 28 weeks were independently associated perinatal with intra-amniotic inflammation. A strategy considering these two non-invasive parameters (either women admitted < 28 weeks or women admitted between mm >/= 28 and < 32 weeks with a cervical length < 15 mm) could detect 84. % of women with intra-amniotic transvaginal inflammation with a positive predictive value of 48.8%, providing improved diagnostic indices compared to either variable considered alone. CONCLUSIONS: Cervical within length and gestational age at admission can be used as a non-invasive method to assess the risk of intra-amniotic inflammation value in preterm labor and intact membranes. Copyright (c) 2009 ISUOG. Published by John Wiley & Sons, Ltd.

Abstract and Virtually nothing is known about disease resistance in solitary bees, so expressed sequence tag (EST) databases were developed to search conserved for immune response genes in the alfalfa leafcutting bee. We identified 104 putative immunity-related genes from both healthy and pathogen-challenged databases bee larvae, and 12 more genes using PCR amplification. The genes identified coded for proteins with a wide variety of resistance innate immune response functions, including pathogen recognition, phagocytosis, the prophenoloxidase cascade, melanization, coagulation and several signalling pathways. Some immune response Abstract genes were highly conserved with honey bee genes, and more distantly related to other insects. The data presented provides the including first analysis of immune function in a solitary bee and provides a foundation for the further analysis of gene expression proteins patterns in bees.

Abstract acid A full-length Hsp83, named SnoHsp83, cDNA from the corn stalk borer, Sesamia nonagrioides, was cloned and sequenced. Genomic analysis showed in that the SnoHsp83 gene is unique. The size of the SnoHsp83 cDNA was found to be approximately 2.6 kb. The sequenced. deduced polypeptide comprised 717 amino acid residues, with a molecular mass of 82.6 kDa. It contained all the highly conserved the amino acid motifs that characterize the cytosolic members of the hsp90 family. We investigated the expression of SnoHsp83 gene in Abstract response to diapause and heat/cold stress. SnoHsp83 is constitutively expressed in non-diapausing larvae and is induced 15-fold by heat. SnoHsp83 the displays a similar pattern to SnoHsc70 under diapause conditions, when extra larval moults occur. Our results indicate that the SnoHsp83 highly gene could be involved in the developmental process that occurs between two moults.

Osmia rates lignaria is a solitary bee that overwinters as a fully-eclosed, cocooned, unfed adult. Our objective is to understand the effect a of wintering temperature on diapause maintenance and termination in this species. We measure respiration rates and weight loss in individuals We exposed to various wintering temperatures ( , 4, 7, 22 degrees C, outdoors) and durations (28, 84, 140, 196, 252 days).objective We use time to emerge and respiration response (respiration rate measured at 22 degrees C) as indicators of diapause intensity.Osmia Adults spontaneously lower their respiration rates to approximately .1ml/g*h within one month after adult eclosion, indicating obligatory diapause. Non-wintered individuals a maintain low respiration rates, but lose weight rapidly and die by mid-winter. In wintered adults, two phases can be distinguished.by First, respiration response undergoes a rapid increase and then reaches a plateau. This phase is similar in bees wintered at lower , 4 and 7 degrees C. In the second phase, respiration response undergoes an exponential increase, which is more pronounced emerge at the warmer temperatures. Composite exponential functions provide a good fit to the observed respiration patterns. Adults whose respiration response which has reached .45ml/g*h emerge promptly when exposed to 20 degrees C, indicating diapause completion. Individuals wintered for short periods do This not reach such respiration levels. When exposed to 20 degrees C these individuals lower their metabolic rate, and their emergence and time is extended. The relationship between respiration rates and emergence time follows a negative exponential function. We propose two alternative an models of diapause termination to interpret these results.

Three organization heat shock protein transcripts, hsp90, hsp70, hsc70, isolated from the corn earworm, Helicoverpa zea, were evaluated for their responsiveness to time. diapause and thermal stress. These Hsps showed high homology to their counterparts in other species. A phylogenetic analysis of the showed Hsp90 sequence was consistent with the known classification of insects. Northern blot hybridization indicated the presence of hsp90 transcripts in Helicoverpa all tissues, but expression in the brain-subesophageal complex was especially pronounced. The genomic organization of hsp90 examined by Southern blots Three suggested the presence of a single copy of hsp90 in the H. zea genome. The expression patterns after heat shock strongly indicated that hsp70 and hsp90 were heat-inducible, although hsp70 was more strongly induced than hsp90, and hsc70 was indeed a The constitutively-expressed member of the hsp70 family. Expression of hsp70 and hsc70 were not altered by the diapause program, but hsp90 consistent was down-regulated at this time. Low temperatures ( -4(o)C) and recovery from low temperature elicited hsp70 and hsp90 responses, but not presence an hsc70 response. Thus, unlike several other species, H. zea does not up-regulate hsp70 during pupal diapause, but the down-regulation by of hsp90 is consistent with the pattern observed in several other species during diapause. Our results also indicate that hsp90 than and hsp70 are responsive to low temperature in both diapausing and nondiapausing pupae.

We arrest propose that a shut-down in expression of ribosomal protein S2 (rpS2) contributes to regulation of diapause in adult females of rpS2 Culex pipiens. While this gene is expressed continuously in nondiapausing females reared under long-day conditions, it is strongly down-regulated 5-18 this days after adult eclosion in females reared under the short-day conditions that induce diapause. The possibility that this shut-down in (rpS2) expression of rpS2 contributes to the arrest in ovarian development characteristic of diapause is bolstered by the diapause-like arrest in We follicle growth observed when nondiapausing females are injected with dsrpS2. A control gene encoding another ribosomal protein, L19, is expressed equally equally in nondiapausing and diapausing females, and RNA interference directed against rpL19 did not arrest follicle growth, thus indicating that nondiapausing the response we observed in knocking down expression of rpS2 is not common to all ribosomal proteins. Diapause in C.in pipiens is readily terminated with juvenile hormone (JH), and in this study we demonstrated that an exogenous application of JHIII short-day can rescue the arrest in follicle growth caused by dsrpS2. Together, these results suggest that rpS2 plays a critical role the in arresting the ovarian development associated with diapause in this mosquito.

Culex with pipiens, the mosquito that vectors West Nile virus in North America, overwinters in an adult diapause (dormancy) that is programmed including by the short day length and low temperatures of autumn. In response to these environmental signals, females cease feeding on autumn. blood and instead seek sources of nectar used to generate the huge lipid reserves required for winter survival. To identify an regulatory networks that regulate fat accumulation and fat consumption during diapause, we compared expression of fat-related genes from nondiapausing females Culex with expression of those same genes in early and late diapause and at diapause termination. Among the 31 genes we the examined, four were expressed more highly in early diapause than in nondiapause, while 14 genes were downregulated in early diapause.expressed In the transition from early to late diapause, 19 genes related to fat metabolism were upregulated. As reported previously, fatty in acid synthase, identified as fas-1 in this study, was upregulated in early diapause. Numerous fat metabolism genes, including multiple kinetic regulatory classes and genes involved in beta-oxidation, an energy-generation step, were suppressed in early diapause but were highly expressed in late this diapause and at diapause termination. RNAi analysis revealed that the fas-1 gene and others (fas-3 and fabp) have important roles from in fat storage during early diapause. When expression of these genes is suppressed, the female mosquitoes fail to sequester the four lipids needed for overwintering. Key words: Diapause, Culex pipiens, Lipid metabolism, RNAi.

Previous facultative analyses of diapause in insects have most often focused on the timing of the switch from non-diapausing to diapausing offspring stage in bivoltine populations and have assumed that diapause is irreversible or that the insect cannot survive winter if not in not diapause. Many insects exhibit more flexibility in their life cycles, such as the age at which diapause begins, and facultative diapausing diapause, that may influence the evolution of different diapause strategies in different environments. The grasshopper Melanoplus sanguinipes F.(Orthoptera: Acrididae),Previous has a very wide geographic range over which diapause characteristics vary greatly. Embryonic diapause in this species may be under degrees maternal control, may be obligate or facultative (i.e., may be averted by cold temperature treatment of pre-diapause embryos), and embryos from may enter diapause at different ages. Diapause traits were examined in two populations of M. sanguinipes from very different environments.be In the population from a temperate climate (Idaho, USA), diapause was facultative, i.e., pre-diapause embryos averted diapause when held at has 5 degrees C for 90 days at all ages tested (7 days and older). The Idaho embryos entered diapause in more. late stage of development if held at 22 degrees C for 30 days or more. In populations from subarctic Alaska,for USA, embryos also entered diapause in a late stage of development, but diapause was obligate and could not be averted M. by chilling in the pre-diapause stages. Simulated evolution of these traits over a wide range of season-lengths showed that late C stage diapause is an essential trait in very short season environments, resulting in early hatching, and a semivoltine life-cycle. Facultative stage diapause enabled bivoltinism to be a viable strategy in shorter seasons than when diapause was obligate. At transitions from semivoltine diapause. to univoltine, and from univoltine to bivoltine life cycles, populations with obligate diapause adopted a strategy of no diapause (via their maternal effects) to enable univoltine life cycles.

The muscles cDNA encoding beta-tubulin in the mosquito Culex pipiens has high similarity with the beta-tubulins reported in other insects. In this while study, we examine expression of this gene and microtubule abundance in relation to diapause and low temperature. While non-diapausing mosquitoes this express beta-tubulin highly in their thoracic muscles, expression is quite low during adult diapause. The abundance of microtubules was also the much lower in flight muscles of diapausing adults than in flight muscles from non-diapausing individuals, as confirmed by laser confocal The microscopy of tubulins stained using indirect immunofluorescence. Low temperatures decreased microtubule abundance in midguts of non-diapausing mosquitoes, but microtubule abundance by in diapausing mosquitoes was already low and remained unchanged by low temperature exposure. Overall, pixel intensity averages were higher in in the flight muscles than in the midguts, and again low temperatures decreased microtubule abundance in the flight muscles of non-diapausing reduced females, while levels remained consistently low in diapausing females. These results clearly indicate that a decrease in microtubule abundance is diapause. evoked both by the programming of diapause and, in non-diapausing females, by exposure to low temperatures. Quite possibly the reduced decreased microtubule abundance in the flight muscles and reduced expression of beta-tubulin are functionally correlated to the reduction in flight activity low that is associated with low temperature and diapause.

The in hsp70 level in the bodies of 1(st) instars of grasshoppers Chorthippus brunneus from unpolluted (Pilica) and polluted (Olkusz, Szopienice) sites larvae and additionally exposed to various doses of zinc during diapause and embryonic development prior to hatching were measured by Western and Blotting. The main aim of our work was to assess the relationship between the age of female grasshoppers originating from (Pilica) variously polluted habitat and the hsp70 level in their progeny. Possible reasons for population variation in hsp70 levels were discussed.The The hsp70 level in the offspring's body depended on the place of origin. The strongest expression of hsp70 was found from in the bodies of larvae hatching from the eggs laid by young females from Pilica (reference site). In contrast, a the low initial level of hsp70 in larvae from polluted sites, especially in young females' progeny, was observed. The application of or zinc during diapause influenced the hsp70 level in grasshopper larvae; however, the direction of the changes depended on the insects'habitat place of origin. In larvae from the reference site, and also (but to a lesser degree) from Olkusz, the increase of in the hsp70 level after zinc treatment was most pronounced. Whereas in grasshoppers from Szopienice, zinc (in 100mug.g(-1) dry weight polluted of sand) did not change the hsp70 level, or (in 500mug.g(-1) dry weight of sand) caused a reduction in hsp70.hatching The differences may result from maternal effects; however, possible adaptation also cannot be excluded. To confirm this statement further studies larvae; are needed.

Survival agar rates of Ascosphaera aggregata and A. apis over the course of a year were tested using different storage treatments. For of spores, the storage methods tested were freeze-drying and ultra-low temperatures, and for hyphae, freeze-drying, agar slants, and two methods of year ultra-low temperatures. Spores of A. aggregata and A. apis stored well at -80 degrees C and after freeze-drying. A. aggregata A. hyphae did not store well under any of the methods tested while A. apis hyphae survived well using cryopreservation. Spores Survival produced from cryopreserved A. apis hyphae were infective. Long-term storage of these two important fungal bee diseases is thus possible.-80