Extended hypoxia in the alfalfa leafcutting bee, Megachile rotundata, increases survival but causes sub-lethal effects.

Many insects are tolerant of hypoxic conditions, but survival may come at a cost to long-term health. The alfalfa leaf-cutting bee, Megachile rotundata, develops in brood cells inside natural cavities, and may be exposed to hypoxic conditions for extended periods of time. Whether M. rotundata is tolerant of hypoxia, and whether exposure results in sub-lethal effects, has never been investigated. Overwintering M. rotundata prepupae were exposed to 10%, 13%, 17%, 21% and 24% O2 for 11 months. Once adults emerged, five indicators of quality - emergence weight, body size, feeding activity, flight performance, and adult longevity, - were measured to determine whether adult bees that survived past exposure to hypoxia were competent pollinators. M. rotundata prepupae are tolerant of hypoxic condition and have higher survival rates in hypoxia, than in normoxia. Under hypoxia, adult emergence rates did not decrease over the 11 months of the experiment. In contrast, bees reared in normoxia had decreased emergence rates by 8 months, and were dead by 11 months. M. rotundata prepupae exposed to extended hypoxic conditions had similar emergence weight, head width, and cross-thorax distance compared to bees reared in standard 21% oxygen. Despite no significant morphological differences, hypoxia-exposed bees had lower feeding rates and shorter adult lifespans. Hypoxia may play a role in post-diapause physiology of M. rotundata, with prepupae showing better survival under hypoxic conditions. Extended exposure to hypoxia, while not fatal, causes sub-lethal effects in feeding rates and longevity in the adults, indicating that hypoxia tolerance comes at a cost.

Thermal history influences diapause development in the solitary bee Megachile rotundata.

Respiration rate, time to pupation and the expression patterns of selected genes were examined during the diapause to post-diapause transition in the alfalfa leafcutting bee, Megachile rotundata held at constant 4 degrees C in winter storage. Respiration quotients were at or below 0.7 from December to May and then increased to over 0.8 in June and July. The time required for prepupae to reach the pupal stage following transfer to 29 degrees C decreased from 23 days in December to 10 days in July. HSP70 was expressed at a consistently high level in all the diapausing prepupae stored at 4 degrees C. In contrast, we demonstrated previously that HSP70 expression in diapausing prepupae maintained under field conditions began decreasing after December and was expressed at trace levels in the June samples. Transferring prepupae stored at 4 to 25 degrees C at monthly intervals from December to July induced a significant decrease in HSP70. Levels of HSC70 showed no changes during the transition to post-diapause development in prepupae maintained at 4 degrees C. Transferring the prepupae to 25 degrees C during the April-June time interval elicited an increase in HSC70 expression. HSP90 was expressed at a consistent level in prepupae stored at 4 degrees C but decreased to very low levels after being transferred to 25 degrees C in December-February prepupae: no decrease was noted in the April-July prepupae. Actin was expressed at trace levels in the diapausing prepupae maintained at 4 degrees C and increased slightly in the post-diapausing pupae. Transferring prepupae stored at 4 to 25 degrees C at monthly intervals from December to July induced an increase in actin expression. These results indicate that the level of gene expression for selected genes in diapausing and post-diapause bees is highly influenced by their thermal history.

Differential gene expression in Perillus bioculatus nymphs fed a suboptimal artificial diet.

Fragments of two artificial diet up-regulated and two prey up-regulated transcripts were isolated from the predatory Pentatomid Perillus bioculatus using suppression subtractive hybridization. A BlastX search found similarities for two diet-upregulated clones, i.e., the tyrosine-3-monooxygenase gene and the gene for the chitin binding protein, Gasp. The probe generated from the tyrosine-3-monooxygenase clone hybridized to two transcripts 2.3 and 1.2kb in size. The two transcripts were differentially regulated: the 2.3kb transcript was upregulated in the first and late third instar diet-fed nymphs, whereas the 1.2kb transcript was upregulated in the second and early third instar diet-fed nymphs. The Gasp gene was upregulated in late third instar nymphs. A positive correlation was found between levels of expression of the isolated genes and the number of generations the insects had been reared on the artificial diet.

Temporal variation in overwintering gene expression and respiration in the solitary bee Megachile rotundata.

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.

Isolation and characterization of three diapause-associated transcripts from the Colorado potato beetle, Leptinotarsa decemlineata.

Using suppressive subtractive hybridization, fragments of three diapause-associated transcripts (DAT-1, 2 and 3) were isolated from the Colorado potato beetle, Leptinotarsa decemlineata. Full-length clones were developed for the transcripts. DAT-1 encodes a deduced protein 286 amino acids in length with limited identity to several proteins with leucine-rich domains. DAT-2 encodes a deduced protein 229 amino acids in length with 27% identity, 40% similarity to the desiccation stress protein from Tenebrio molitor. DAT-3 encodes a deduced protein 97 amino acids in length with identity to no known protein. DAT-1 and 2 have similar expression patterns as determined by northern blot analysis. Trace levels of these two transcripts are first detected in 3-day-old diapause-programmed adults with a significant increase in expression on day 6. Expression of DAT-3 begins on day 12 in diapause-programmed adults and expression levels increase until the beetles enter diapause. Expression of DAT-1, 2 and 3 continues at least 60 days into diapause.

Differential expression of two HSP70 transcripts in response to cold shock, thermoperiod, and adult diapause in the Colorado potato beetle.

Partial clones for two members of Leptinotarsa decemlineata inducible 70kDa heat shock protein family (LdHSP70A and B) were developed using RT-PCR. LdHSP70A, but not LdHSP70B, was upregulated during adult diapause. The ability of L. decemlineata to express these two genes in response to subzero temperatures depended on the thermal history of the beetles. Chilling diapausing beetles increased the rate at which both LdHSP70A and B were expressed following a cold shock at -10 degrees C. Following cold shock at -10 degrees C, LdHSP70B expression peaked after 3h at 15 degrees C for chilled diapausing individuals, decreasing to near background levels by the sixth hour. In contrast, nonchilled diapausing beetles expressed their highest level of LdHSP70B only after 6h at 15 degrees C. Diapausing beetles exposed to a thermoperiod with a mean temperature of either 0 or -2.5 degrees C expressed significantly higher levels of both LdHSP70A and B than beetles exposed to constant 0 or -2.5 degrees C. These results demonstrate that the expression of LdHSP70A and B is differentially regulated in response to diapause and environmental conditioning.

Developmental upregulation of inducible hsp70 transcripts, but not the cognate form, during pupal diapause in the flesh fly, Ssarcophaga crassipalpis.

Partial clones of the Sarcophaga crassipalpis heat shock protein 70 (hsp70) and of heat shock cognate 70 (hsc70) were developed by RT-PCR and library screening respectively. These clones were used to probe total RNA northern blots for the expression of transcripts in response to high and low temperature stress and in conjunction with the entry into an overwintering pupal diapause. In nondiapausing individuals, hsp70 was highly expressed in response to a 40 degrees C heat shock, while hsc70 was unaffected by the heat stress. In contrast, both hsp70 and hsc70 were upregulated in nondiapausing flies following a -10 degrees C cold shock. In diapausing pupae, hsp70 was highly upregulated during diapause, even at a non-stress temperature of 20 degrees C. Upregulation was initiated at the onset of diapause and persisted throughout diapause. During diapause, heat shock did not further elevate the level of hsp70 expression. Within 12 h after diapause was terminated, hsp70 ceased to be expressed. The expression of hsc70 was unaltered by diapause. The developmental regulation of hsp70 in relation to diapause suggests a critical role for this stress protein during insect dormancy.

Upregulation of a 23 kDa small heat shock protein transcript during pupal diapause in the flesh fly, Sarcophaga, crassipalpis.

A diapause upregulated cDNA clone was isolated from a cDNA library generated from brain mRNA of diapausing Sarcophaga crassipalpis pupae. The clone hybridized to a 1600 bp transcript on a northern blot. The insert is 823 bp in length, has a tentative open reading frame of 615 bp, and codes for a 23 kDa protein. The clone has a high level of identity at the amino acid level with the four small heat shock proteins of Drosophila melanogaster. Northern analysis revealed no detectable expression of the transcript in diapause- or nondiapause-programmed wandering larvae, and only trace expression in nondiapausing pupae. But, the transcript was highly expressed beginning at the onset of diapause and continuing throughout diapause. Expression promptly decreased when diapause was terminated. In nondiapausing individuals the transcript was highly expressed in response to cold shock or heat shock, but temperature stress did not cause greater expression in diapausing pupae. The results imply that expression of this small heat shock protein, a response elicited by temperature stress in nondiapausing individuals, is a normal component of the diapause syndrome. The upregulation of this gene during diapause suggests that it plays an essential role during this overwintering developmental arrest.

Diapause-specific gene expression in pupae of the flesh fly Sarcophaga crassipalpis.

Several cDNAs isolated from brains of diapausing pupae of the flesh fly, Sarcophaga crassipalpis, show expression patterns unique to diapause. To isolate such cDNAs a diapause pupal brain cDNA library was screened by using an elimination hybridization technique, and cDNAs that did not hybridize with cDNA probes constructed from the RNA of nondiapausing pupae were selected for further screening. The 95 clones that did not hybridize in the initial library screen were selected for further characterization. These clones were then screened against diapause and nondiapause pupal poly(A)+ Northern blots. The secondary screen identified 4 diapause-up-regulated clones, 7 diapause-down-regulated clones, 8 clones expressed equally in both diapause and nondiapause, and 75 clones without detectable expression. The diapause-up-regulated and down-regulated clones were further characterized by partial DNA sequencing and identity searches by using GenBank. Identities between our cloned cDNAs and other genes included those linked to cell cycle progression, stress responses, and DNA repair processes. The results suggest that insect diapause is not merely a shutdown of gene expression but is a unique, developmental pathway characterized by the expression of a novel set of genes.