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

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 degrees C revealed no differences among respiration rates of initially diapausing and long-term stored larvae. A 15 degrees increase in temperature elevated respiration in both diapausing and stored larvae, with levels of CO(2) release ranging between 8- and 14-fold higher at 20 degrees C than at 5 degrees C. Similarly, 6-10-fold increases in O(2) 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.

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

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

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

Osmia lignaria is a solitary bee that overwinters as a fully-eclosed, cocooned, unfed adult. Our objective is to understand the effect of wintering temperature on diapause maintenance and termination in this species. We measure respiration rates and weight loss in individuals exposed to various wintering temperatures (0, 4, 7, 22 degrees C, outdoors) and durations (28, 84, 140, 196, 252 days). We use time to emerge and respiration response (respiration rate measured at 22 degrees C) as indicators of diapause intensity. Adults spontaneously lower their respiration rates to approximately 0.1ml/g*h within one month after adult eclosion, indicating obligatory diapause. Non-wintered individuals maintain low respiration rates, but lose weight rapidly and die by mid-winter. In wintered adults, two phases can be distinguished. First, respiration response undergoes a rapid increase and then reaches a plateau. This phase is similar in bees wintered at 0, 4 and 7 degrees C. In the second phase, respiration response undergoes an exponential increase, which is more pronounced at the warmer temperatures. Composite exponential functions provide a good fit to the observed respiration patterns. Adults whose respiration response has reached 0.45ml/g*h emerge promptly when exposed to 20 degrees C, indicating diapause completion. Individuals wintered for short periods do not reach such respiration levels. When exposed to 20 degrees C these individuals lower their metabolic rate, and their emergence time is extended. The relationship between respiration rates and emergence time follows a negative exponential function. We propose two alternative models of diapause termination to interpret these results.

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

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

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

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

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

Receptor binding techniques have been widely used in different biochemical applications, isolated membranes being the most used receptor preparation in this type of assays. In this study, intact cells were compared to isolated membranes as receptor support for radioligand receptor-binding assay. Growth hormone secretagogue receptor 1a (GHSR-1a) expressed in HEK293 cells was used as a model of G protein-coupled receptors. Differences between using intact cells in suspension or isolated membranes were evaluated for different aspects of the receptor binding assay: total binding variations while both receptor preparations remain on ice, modifications in incubation conditions, saturation and competition using different agonists. Intact cells are more prone to variability. While under optimized settings both preparation were equivalent, the K(d) value for intact cells was three times higher than using isolated membranes. However, no significant differences were observed in competition assays obtaining practically identical K(i) values for all ligands tested. For the GHSR-1a receptor, isolated membranes are the better choice if particular incubation conditions are required (less variability) while intact cells yield easy, fast and physiological conditions for receptor binding assay.

One hundred seventy human uropathogenic Escherichia coli (UPEC) clinical isolates were compared with 35 E. coli strains isolated from feces of a control group to determine the presence of the set1, sen and astA genes encoding the ShET-1, ShET-2, and EAST toxins, respectively. Overall, 27 (16%), 8 (8%) and 0 UPEC isolates presented the set1B, the astA, and the sen genes, respectively. This is the first time the set gene has been found in UPEC clinical isolates.

The thermotolerance of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), was evaluated by subjecting larvae and prepupae to a number of time-temperature regimes. Three independent experiments were conducted during 2006 and 2007 by heating emerald ash borer infested firewood in laboratory ovens. Heat treatments were established based on the internal wood temperature. Treatments ranged from 45 to 65 degrees C for 30 and 60 min, and the ability of larvae to pupate and emerge as adults was used to evaluate the success of each treatment. A fourth experiment was conducted to examine heat treatments on exposed prepupae removed from logs and subjected to ambient temperatures of 50, 55, and 60 degrees C for 15, 30, 45, and 60 min. Results from the firewood experiments were consistent in the first experiment. Emergence data showed emerald ash borer larvae were capable of surviving a temperatures-time combination up to 60 degrees C for 30 min in wood. The 65 degrees C for 30 min treatment was, however, effective in preventing emerald ash borer emergence on both dates. Conversely, in the second experiment using saturated steam heat, complete mortality was achieved at 50 and 55 degrees C for both 30 and 60 min. Results from the prepupae experiment showed emerald ash borer survivorship in temperature-time combinations up to 55 degrees C for 30 min, and at 50 degrees C for 60 min; 60 degrees C for 15 min and longer was effective in preventing pupation in exposed prepupae. Overall results suggest that emerald ash borer survival is variable depending on heating conditions, and an internal wood temperature of 60 degrees C for 60 min should be considered the minimum for safe treatment for firewood.

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

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

Osmia lignaria is a solitary bee that overwinters as a fully-eclosed, cocooned, unfed adult. Our objective is to understand the effect of wintering temperature on diapause maintenance and termination in this species. We measure respiration rates and weight loss in individuals exposed to various wintering temperatures (0, 4, 7, 22 degrees C, outdoors) and durations (28, 84, 140, 196, 252 days). We use time to emerge and respiration response (respiration rate measured at 22 degrees C) as indicators of diapause intensity. Adults spontaneously lower their respiration rates to approximately 0.1ml/g*h within one month after adult eclosion, indicating obligatory diapause. Non-wintered individuals maintain low respiration rates, but lose weight rapidly and die by mid-winter. In wintered adults, two phases can be distinguished. First, respiration response undergoes a rapid increase and then reaches a plateau. This phase is similar in bees wintered at 0, 4 and 7 degrees C. In the second phase, respiration response undergoes an exponential increase, which is more pronounced at the warmer temperatures. Composite exponential functions provide a good fit to the observed respiration patterns. Adults whose respiration response has reached 0.45ml/g*h emerge promptly when exposed to 20 degrees C, indicating diapause completion. Individuals wintered for short periods do not reach such respiration levels. When exposed to 20 degrees C these individuals lower their metabolic rate, and their emergence time is extended. The relationship between respiration rates and emergence time follows a negative exponential function. We propose two alternative models of diapause termination to interpret these results.

Three heat shock protein transcripts, hsp90, hsp70, hsc70, isolated from the corn earworm, Helicoverpa zea, were evaluated for their responsiveness to diapause and thermal stress. These Hsps showed high homology to their counterparts in other species. A phylogenetic analysis of the Hsp90 sequence was consistent with the known classification of insects. Northern blot hybridization indicated the presence of hsp90 transcripts in all tissues, but expression in the brain-subesophageal complex was especially pronounced. The genomic organization of hsp90 examined by Southern blots suggested the presence of a single copy of hsp90 in the H. zea genome. The expression patterns after heat shock indicated that hsp70 and hsp90 were heat-inducible, although hsp70 was more strongly induced than hsp90, and hsc70 was indeed a constitutively-expressed member of the hsp70 family. Expression of hsp70 and hsc70 were not altered by the diapause program, but hsp90 was down-regulated at this time. Low temperatures (0-4(o)C) and recovery from low temperature elicited hsp70 and hsp90 responses, but not an hsc70 response. Thus, unlike several other species, H. zea does not up-regulate hsp70 during pupal diapause, but the down-regulation of hsp90 is consistent with the pattern observed in several other species during diapause. Our results also indicate that hsp90 and hsp70 are responsive to low temperature in both diapausing and nondiapausing pupae.

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

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

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

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

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