A nondiapausing variant of the flesh fly, Sarcophaga bullata, that shows arrhythmic adult eclosion and elevated expression of two circadian clock genes, period and timeless

We describe a variant of the flesh fly, Sarcophaga bullata, which fails to enter pupal diapause in response to short daylength and low temperatures. This fly also has an arrhythmic adult eclosion pattern: rather than eclosing in early photophase, the variant ecloses arrhythmically throughout the photophase and scotophase. The loss of both diapause (photoperiodic response) and the gating of adult eclosion (presumably a circadian response) suggests that the same clock system is involved in these two responses. An examination of the expression patterns of the clock genes period and timeless demonstrates that both genes are present in the nondiapausing variant, but surprisingly, both genes are expressed at higher levels. This abnormality we observe, possibly the consequence of an upstream clock gene malfunction or a malfunction of the autoregulatory loop, results in disruption of a component of the clock system that is apparently needed for both photoperiodism and circadian rhythmicity.     

Calls,colours, shape,and genes: a multi‐trait approach to the study of geographic variation in the Amazonian frog Allobates femoralis

Evolutionary divergence in behavioural traits related to mating may represent the initial stage of speciation. Direct selective forces are usually invoked to explain divergence in mate‐recognition traits, often neglecting a role for neutral processes or concomitant differentiation in ecological traits. We adopted a multi‐trait approach to obtain a deeper understanding of the mechanisms behind allopatric divergence in the Amazonian frog, Allobates femoralis. We tested the null hypothesis that geographic distance between populations correlates with genetic and phenotypic divergence, and compared divergence between mate‐recognition (acoustic) and ecological (coloration, body‐shape) traits. We quantified geographic variation in 39 phenotypic traits and a mitochondrial DNA marker among 125 individuals representing eight populations. Geographic variation in acoustic traits was pronounced and tracked the spatial genetic variation, which appeared to be neutral. Thus, the evolution of acoustic traits tracked the shared history of the populations, which is unexpected for pan‐Amazonian taxa or for mate‐recognition traits. Divergence in coloration appeared uncorrelated with genetic distance, and might be partly attributed to local selective pressures, and perhaps to Batesian mimicry. Divergence in body‐shape traits was low. The results obtained depict a complex evolutionary scenario and emphasize the importance of considering multiple traits when disentangling the forces behind allopatric divergence. ©2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 826–838.     

Dehydration-induced cross tolerance of Belgica antarctica larvae to cold and heat is facilitated by trehalose accumulation

Larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae), are frequently exposed to dehydrating conditions on the Antarctic Peninsula. In this study, we examined how rates and levels of dehydration alter heat and cold tolerance and how these relate to levels of trehalose within the insect. When dehydrated, larvae tolerated cold and heat stress more effectively, although resistance to cold was more pronounced than heat resistance. Slow dehydration was more effective than rapid dehydration in increasing temperature tolerance. Severe dehydration (50% reduction in water content) caused a much greater increase in temperature tolerance than did mild dehydration (e.g. 10% water loss). Larvae severely dehydrated at a slow rate (98% RH) were more temperature tolerant than those dehydrated quickly (0 or 75% RH). These results indicate that the slower dehydration rate allows the larvae to more effectively respond to reduced water levels and that physiological adjustments to desiccation provide cross tolerance to cold and heat. Levels of trehalose increased during dehydration and are likely a major factor increasing subsequent cold and heat resistance. This hypothesis was also supported by experimental results showing that injection of trehalose enhanced resistance to temperature stress and dehydration. We conclude that changes in temperature tolerance in B. antarctica are linked to the rate and severity of dehydration and that trehalose elevation is a probable mechanism enhancing this form of cross tolerance.     

A potential role for ribosomal protein S2 in the gene network regulating reproductive diapause in the mosquito Culex pipiens

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.     

Evolution of base composition in the insulin and insulin-like growth factor genes

The genomes of homeothermic (warm-blooded) vertebrates are mosaic interspersions of homogeneously GC-rich and GC-poor regions (isochores). Evolution of genome compartmentalization and GC-rich isochores is hypothesized to reflect either selective advantages of an elevated GC content or chromosome location and mutational pressure associated with the timing of DNA replication in germ cells. To address the present controversy regarding the origins and maintenance of isochores in homeothermic vertebrates, newly obtained as well as published nucleotide sequences of the insulin and insulin-like growth factor (IGF) genes, members of a well-characterized gene family believed to have evolved by repeated duplication and divergence, were utilized to examine the evolution of base composition in nonconstrained (flanking) and weakly constrained (introns and fourfold degenerate sites) regions. A phylogeny derived from amino acid sequences supports a common evolutionary history for the insulin/IGF family genes. In cold- blooded vertebrates, insulin and the IGFs were similar in base composition. In contrast, insulin and IGF-II demonstrate dramatic increases in GC richness in mammals, but no such trend occurred in IGF- I. Base composition of the coding portions of the insulin and IGF genes across vertebrates correlated (r = 0.90) with that of the introns and flanking regions. The GC content of homologous introns differed dramatically between insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level of noncoding regions in neighboring genes. Our findings suggest that the base composition of introns and flanking regions is determined by chromosomal location and the mutational pressure of the isochore in which the sequences are embedded. An elevated GC content at codon third positions in the insulin and the IGF genes may reflect selective constraints on the usage of synonymous codons.      

Preventing insect diapause with hormones and cholera toxin

The decision to enter pupal diapause can be reversed in the flesh fly $\text{Sarcophaga crassipalpis}$ by treatment with a juvenile hormone analogue, ecdysterone, or cholera toxin. A topical application of juvenile hormone analogue (10μg) is effective in preventing diapause if applied to newly ecdysed 3rd instar larvae. An ecdysterone injection (1μg) into post-fed 3rd instar larvae can avert diapause, and even lower dosages (0.01μg) are effective if injected into young pupae immediately before the onset of diapause. Cholera toxin, a stimulant of adenylate cyclase, can prevent diapause if 1μg is injected into larvae 24 hrs. prior to pupariation. This is the first report of an effect of cholera toxin on insects. A 2-day exposure to 33°C was the only physical manipulation shown to be capable of reversing the previous commitment to enter pupal diapause.     

Eclosion behavior in tsetse (Diptera: Glossinidae): Extrication from the puparium and expansion of the adult

The tsetse adult extricates itself from the puparium and surrounding substrate by a series of muscular contractions that generate a Stereotypic pattern of changes in hemolymph pressure. The digging action of the fly can be distinguished from a second pattern of hemocoelic pulsations that is used to remove obstacles from its path. When the fly is restrained extrication behavior will persist for over 10 h. If the adult's legs are freed while the remainder of the body remains encased in the puparium, the fly fails to engage in extrication behavior, a result which suggests that freedom of the legs switches off extrication behavior and permits the onset of expansion of the body to its final adult size and shape. Expansion behavior includes walking, grooming, pumping air into the gut, and contracting the abdominal muscles to generate rhythmic pulses of hemocoelic pressure. A barographic record of internal pressure changes reflects the dynamics of this morphogenetic process. Results from tsetse are compared with previous observations recorded in flesh flies.     

Sequence and transcription patterns of 60S ribosomal protein P0, a diapause-regulated AP endonuclease in the flesh fly, Sarcophaga crassipalpis

We have isolated and sequenced a 1308bp clone from a pupal brain cDNA library of the flesh fly, Sarcophaga crassipalpis, showing 97% amino acid (aa) sequence similarity to Ceratitis capitata 60S acidic ribosomal protein P0 (CcP0) and 93% aa sequence similiarity to Drosophila melanogaster P0 (DmP0). DmP0 is a multifunctional protein necessary for efficient protein translation of the 60S ribosome as well as DNA repair via AP3 endonuclease activity. In this study, we observed that S. crassipalpis P0 (ScP0) is cyclically regulated throughout the fly's overwintering pupal diapause. Expression of ScP0 cycles out of phase with the 4day cycles of O(2) consumption: the peak day of O(2) consumption is characterized by low ScP0 expression, while high expression is noted during the trough of the O(2) consumption cycle. The O(2) cycles, which are in turn driven by cycles of juvenile hormone (JH), can be eliminated by application of a JH analog (JHA). Pupae rendered acyclic with a JHA application consume O(2) at a constant high rate and ScP0 is consistently downregulated. Our findings thus suggest that the cyclic nature of ScP0 regulation during pupal diapause is linked to the JH-mediated metabolic cycles characteristic of this species.     

Cold hardiness of the fly pupal parasitoid Nasonia vitripennis is enhanced by its host Sarcophaga crassipalpis

Supercooling points (SCPs) and low temperature survival were determined for diapausing and nondiapausing larvae of the ectoparasitoid Nasonia vitripennis. Neither nondiapausing nor diapausing larvae could survive tissue freezing. The SCP profiles were nearly identical for nondiapause-destined (-27 degrees C) and diapausing larvae (-25 degrees C), but these values were not indicative of the lower limits of tolerance in either type of larvae: larvae were killed by chilling at temperatures well above the SCP. Diapausing larvae could withstand low temperature exposures 3-8 times longer than their nondiapausing counterparts. Low temperature survival was enhanced in diapausing and nondiapausing larvae by their encasement within the puparium of the host flesh fly, SARCOPHAGA CRASSIPALPIS: the LT(50)s determined for nondiapausing and diapausing larvae enclosed by fly puparia were 2-3 times higher than values calculated for larvae removed from the puparia. Additional low temperature protection was gained through acquisition of host cryoprotectants during larval feeding: nondiapausing parasitoid larvae that fed on diapausing flesh fly pupae with high levels of glycerol were able to survive exposure to a subzero temperature 4-9 times longer than wasps reared on nondiapausing fly pupae that contained lower quantities of glycerol. Alanine may also contribute to the cold hardiness of N. vitripennis, as evidenced by the fact that larvae feeding on diapausing fly pupae both contained higher concentrations of alanine and exhibited greater cold hardiness. The results thus demonstrate that several critical features of cold hardiness in the wasp are derived from biochemical and physical attributes of the host.