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Phenotypic and genetic analysis of Clock, a new circadian rhythm mutant in Drosophila melanogaster.

Clock is a semidominant X-linked mutation that results in shortening the period of Drosophila melanogaster's free-running locomotor activity rhythm from ca. 24.0 to ca. 22.5 hr. This mutation similarly shortened the phase response curve, determined by resetting activity rhythms with light pulses. Eclosion peaks for Clk cultures were separated by only 22.5 hr instead of the normal 24 hr. Clk was mapped close to, but separable from, another rhythm mutation--period01--by recombination. The estimated distance between these two mutations was short enough to suggest that Clk could be a per allele. If this is the case, the new mutant is unique in that it, unlike other per variants, is associated with essentially normal 1-min courtship song rhythms when Clk is expressed in males. Also, the new rhythm variant could not, in contrast to a short-period per mutation, have its effects on free-running activity rhythms uncovered by deletions. This result, and the lack of coverage of Clk's effects by duplications, suggest that it is not a simple hypomorphic or amorphic mutation.

Animals↗

Genetic control of pheromones in Drosophila simulans. II. kété, a locus on the X chromosome.

The production of Drosophila cuticular hydrocarbons, including contact pheromones, is under polygenic control. To investigate X-linked loci, EMS mutations were induced in Drosophila simulans flies. A mutant strain was discovered which in both sexes show a reduction in the biosynthesis of both 7-tricosene (7-T) the species contact pheromone and all other linear hydrocarbons. The locus controlling this effect, kété, is recessive and was localized to I, 18.5. Unlike a previously identified gene on the second chromosome of this species, Ngbo, kété does not affect the ratio of 7-T:7-pentacosene (7-P). Other reproductive characteristics are also affected, including egg-hatching. However, courtship behaviors in both sexes appear normal.

Animals↗

Genetic and molecular analysis of the X chromosomal region 14B17-14C4 in Drosophila melanogaster: loss of function in NONA, a nuclear protein common to many cell types, results in specific physiological and behavioral defects.

We have performed a genetic analysis of the 14C region of the X chromosome of Drosophila melanogaster to isolate loss of function alleles of no-on-transient A (nonA; 14C1-2; 1-52.3). NONA is a nuclear protein common to many cell types, which is present in many puffs on polytene chromosomes. Sequence data suggest that the protein contains a pair of RNA binding motifs (RRM) found in many single-strand nucleic acid binding proteins. Hypomorphic alleles of this gene, which lead to aberrant visual and courtship song behavior, still contain normally distributed nonA RNA and NONA protein in embryos, and in all available alleles NONA protein is present in puffs of third instar larval polytene chromosomes. We find that complete loss of this general nuclear protein is semilethal in hemizygous males and homozygous cell lethal in the female germline. Surviving males show more extreme defects in nervous system function than have been described for the hypomorphic alleles. Five other essential genes that reside within this region have been partially characterized.

Alleles↗

Genetics of a difference in male cuticular hydrocarbons between two sibling species, Drosophila simulans and D. sechellia.

In seven of the eight species of the Drosophila melanogaster group, the predominant cuticular hydrocarbon of males is 7-tricosene, but in the island endemic species D. sechellia it is 6-tricosene. The phylogeny of the group implies that the novel hydrocarbon profile of D. sechellia is a derived character. Genetic analysis of hybrids between D. sechellia and its close relative D. simulans show that each of the five major chromosome arms carries at least one gene affecting the ratio of the two tricosene isomers, with the right arm of the third chromosome having the largest effect. The species difference in this character is therefore polygenic with the effects of the different chromosome arms generally additive, although there is some epistasis among third-chromosome genes. Observations of courtship by males who have been coated with foreign hydrocarbons suggest that a male's hydrocarbon profile may slightly affect the degree of sexual isolation in one of the reciprocal hybridizations between these species, but that this role is small compared to that played by hydrocarbon differences between females.

Alkenes↗

Geographic structure of mitochondrial and nuclear gene polymorphisms in Australian green turtle populations and male-biased gene flow.

The genetic structure of green turtle (Chelonia mydas) rookeries located around the Australian coast was assessed by (1) comparing the structure found within and among geographic regions, (2) comparing microsatellite loci vs. restriction fragment length polymorphism analyses of anonymous single copy nuclear DNA (ascnDNA) loci, and (3) comparing the structure found at nuclear DNA markers to that of previously analyzed mitochondrial (mtDNA) control region sequences. Significant genetic structure was observed over all regions at both sets of nuclear markers, though the microsatellite data provided greater resolution in identifying significant genetic differences in pairwise tests between regions. Inferences about population structure and migration rates from the microsatellite data varied depending on whether statistics were based on the stepwise mutation or infinite allele model, with the latter being more congruent with geography. Estimated rates of gene flow were generally higher than expected for nuclear DNA (nDNA) in comparison to mtDNA, and this difference was most pronounced in comparisons between the northern and southern Great Barrier Reef (GBR). The genetic data combined with results from physical tagging studies indicate that the lack of nuclear gene divergence through the GBR is likely due to the migration of sGBR turtles through the courtship area of the nGBR population, rather than male-biased dispersal. This example highlights the value of combining comparative studies of molecular variation with ecological data to infer population processes.

Alleles↗

Aberrant splicing and altered spatial expression patterns in fruitless mutants of Drosophila melanogaster.

The fruitless (fru) gene functions in Drosophila males to establish the potential for male sexual behaviors. fru encodes a complex set of sex-specific and sex-nonspecific mRNAs through the use of multiple promoters and alternative pre-mRNA processing. The male-specific transcripts produced from the distal (P1) fru promoter are believed to be responsible for its role in specifying sexual behavior and are only expressed in a small fraction of central nervous system (CNS) cells. To understand the molecular etiology of fruitless mutant phenotypes, we compared wild-type and mutant transcription patterns. These experiments revealed that the fru(2), fru(3), fru(4), and fru(sat) mutations, which are due to P-element inserts, alter the pattern of sex-specific and sex-nonspecific fru RNAs. These changes arise in part from the P-element insertions containing splice acceptor sites that create alternative processing pathways. In situ hybridization revealed no alterations in the locations of cells expressing the P1-fru-promoter-derived transcripts in fru(2), fru(3), fru(4), and fru(sat) pharate adults. For the fru(1) mutant (which is due to an inversion breakpoint near the P1 promoter), Northern analyses revealed no significant changes in fru transcript patterns. However, in situ hybridization revealed anomalies in the level and distribution of P1-derived transcripts: in fru(1) males, fewer P1-expressing neurons are found in regions of the dorsal lateral protocerebrum and abdominal ganglion compared to wild-type males. In other regions of the CNS, expression of these transcripts appears normal in fru(1) males. The loss of fruitless expression in these regions likely accounts for the striking courtship abnormalities exhibited by fru(1) males. Thus, we suggest that the mutant phenotypes in fru(2), fru(3), fru(4), and fru(sat) animals are due to a failure to appropriately splice P1 transcripts, whereas the mutant phenotype of fru(1) animals is due to the reduction or absence of P1 transcripts within specific regions of the CNS.

Amino Acid Sequence↗

Molecular genetic dissection of the sex-specific and vital functions of the Drosophila melanogaster sex determination gene fruitless.

A multibranched hierarchy of regulatory genes controls all aspects of somatic sexual development in Drosophila melanogaster. One branch of this hierarchy is headed by the fruitless (fru) gene and functions in the central nervous system, where it is necessary for male courtship behavior as well as the differentiation of a male-specific abdominal structure, the muscle of Lawrence (MOL). A preliminary investigation of several of the mutations described here showed that the fru gene also has a sex-nonspecific vital function. The fru gene produces a complex set of transcripts through the use of four promoters and alternative splicing. Only the primary transcripts produced from the most distal (P1) promoter are sex-specifically spliced under direction of the sex-determination hierarchy. We have analyzed eight new fru mutations, created by X-ray mutagenesis and P-element excision, to try to gain insight into the relationship of specific transcript classes to specific fru functions. Males that lack the P1-derived fru transcripts show a complete absence of sexual behavior, but no other defects besides the loss of the MOL. Both males and females that have reduced levels of transcripts from the P3 promoter develop into adults but frequently die after failing to eclose. Analysis of the morphology and behavior of adult escapers showed that P3-encoded functions are required for the proper differentiation and eversion of imaginal discs. Furthermore, the reduction in the size of the neuromuscular junctions on abdominal muscles in these animals suggests that one of fru's sex-nonspecific functions involves general aspects of neuronal differentiation. In mutants that lack all fru transcripts as well as a small number of adjacent genes, animals die at an early pupal stage, indicating that fru's function is required only during late development. Thus, fru functions both in the sex-determination regulatory hierarchy to control male sexual behavior through sex-specific transcripts and sex-nonspecifically to control the development of imaginal discs and motorneuronal synapses during adult development through sex-nonspecific transcript classes.

Alleles↗

Technical knockout, a Drosophila model of mitochondrial deafness.

Mutations in mtDNA-encoded components of the mitochondrial translational apparatus are associated with diverse pathological states in humans, notably sensorineural deafness. To develop animal models of such disorders, we have manipulated the nuclear gene for mitochondrial ribosomal protein S12 in Drosophila (technical knockout, tko). The prototypic mutant tko(25t) exhibits developmental delay, bang sensitivity, impaired male courtship, and defective response to sound. On the basis of a transgenic reversion test, these phenotypes are attributable to a single substitution (L85H) at a conserved residue of the tko protein. The mutant is hypersensitive to doxycyclin, an antibiotic that selectively inhibits mitochondrial protein synthesis, and mutant larvae have greatly diminished activities of mitochondrial redox enzymes and decreased levels of mitochondrial small-subunit rRNA. A second mutation in the tko gene, Q116K, which is predicted to impair the accuracy of mitochondrial translation, results in the completely different phenotype of recessive female sterility, based on three independent transgenic insertions. We infer that the tko(25t) mutant provides a model of mitochondrial hearing impairment resulting from a quantitative deficiency of mitochondrial translational capacity.

Animals↗

The green anole (Anolis carolinensis): a reptilian model for laboratory studies of reproductive morphology and behavior.

The green anol (Anolis carolinensis) is an excellent reptilian model for studying reproductive behavior and the neural and muscular morphology that supports it. This lizard has been the subject of behavioral and ecological study for more than 100 yr, and a rich literature exists on its natural history. Both courtship and copulatory behaviors reveal sex and seasonal differences, which allow for the study of mechanisms regulation naturally occurring variation in performance at multiple levels within a single animal model. Green anoles are readily obtained due to their abundance in the wild; once in the laboratory, they are easily maintained, bred, and reared. Background on the natural history and husbandry of this lizard is provided, and the authors' research program on the regulation of reproductive anatomy and behavior is reviewed, Discussion includes the similarities and differences in the mechanisms mediating both structure and function compared with more traditional animal models. This type of comparative research will make it possible to identify the fundamental principles governing reproductive biology, thus advancing both basic and applied knowledge.

Animal Husbandry↗

Strong reproductive skew among males in the multiply mated swordtail Xiphophorus multilineatus (Teleostei).

Male swordtails in the genus Xiphophorus display a conspicuous ventral elongation of the caudal fin, the sword, which arose through sexual selection due to female preference. Females mate regularly and are able to store sperm for at least 6 months. If multiple mating is frequent, this would raise the intriguing question about the role of female choice and male-male competition in shaping the mating system of these fishes. Size-dependent alternate mating strategies occur in Xiphophorus; one such strategy is courtship with a sigmoid display by large dominant males, while the other is gonopodial thrusting, in which small subordinate males sneak copulations. Using microsatellite markers, we observed a frequency of multiple paternity in wild-caught Xiphophorus multilineatus in 28% of families analyzed, but the actual frequency of multiple mating suggested by the correction factor PrDM was 33%. The number of fathers contributing genetically to the brood ranged from one to three. Compared to other species in the family Poeciliidae, both frequency and degree of multiple paternity were low. Paternity was found to be highly skewed, with one male on average contributing more than 70% to the offspring. Hence in this Xiphophorus mating system, typically one male dominates and sneaker males do not appear to be particularly effective. Postcopulatory mechanisms, however, such as sperm competition, are also indicated by our data, using sex-linked phenotypes among the offspring.

Animals↗

Laboratory study of competition between United States strains of Aedes albopictus and Aedes aegypti (Diptera: Culicidae).

Mosquito abatement workers in Houston, Tex., and New Orleans, La., have observed that the recent introduction of Aedes albopictus (Skuse) has been accompanied by a decline and virtual disappearance of Ae. aegypti (L.). This suggests competitive displacement but contradicts the direction of displacement observed in native habitats of Ae. albopictus. The paper reports a series of laboratory studies on competition between U.S. strains of the two species. Larval survivorship, size, and development time were monitored in increasing densities of pure and mixed cultures of the two species under limited and optimal diets. Oviposition preferences were examined by offering gravid females cups containing first or fourth instars of either species. Ethological isolation was studied by simultaneously offering virgin females of one species to males of both species and monitoring courtship behavior as well as rates of oviposition, percentage of hatch, and embryonation. The results of the larval competition studies suggest that interspecific competition increased development time only slightly and had no effect on survival of Ae. albopictus. Competition increased survival in Ae. aegypti. Size of both species was influenced by interspecific competition, but the magnitude and direction of the effect was inconsistent between strains and diets such that neither species emerged consistently larger. The results of the oviposition study indicated a higher fecundity in Ae. aegypti but otherwise demonstrated that both species oviposit without regard to the presence, age, or species of larvae in the oviposition container. The ethological isolation tests demonstrated that Ae. aegypti males were more sexually aggressive than males or Ae. albopictus. However, Ae. albopitus females placed with increasing densities of Ae. aegypti males demonstrated no decrease in rates of oviposition, and eggs laid showed equal rates of hatch and embryonation. In general, the results do not suggest that the U.S Ae. albopictus population is inherently more competitive in the laboratory than Ae. aegypti. Other reasons for the observed decline in Ae. aegypti in the United States are discussed.

Aedes↗

Time-activity budget of adult screwworm behavior (Diptera: Calliphoridae).

A time-activity budget of adult screwworm behavior was determined in an outdoor screened enclosure. Screwworms spent most (75.7%) of their time in the passive behaviors of resting and grooming. The adults spent 9.5% of daytime activity feeding, mainly on flowers or nectaries, but also on animal wound fluids and feces. Adults moving in vegetation preferred walking over flying in both frequency and duration. Flying was a frequent activity, but flights were of short (13.7 s) duration. Courtship, mating, and oviposition constituted less than 1% of a fly's total activity. The unusual behavior characterized as "bubbling" was interpreted as either thermoregulation or a nectar concentration.

Animals↗

Unmelanized plumage patterns in Old World leaf warblers do not correspond to sequence variation at the melanocortin-1 receptor locus (MC1R).

Evolutionary changes in patterns and coloration of plumage are likely to represent a major mechanism for speciation among birds, yet the molecular basis for such changes remains poorly understood. Recently much attention has focused on the melanocortin-1 receptor (MC1R) as a candidate locus for determining the level and extent of epidermal melanin deposition. We tested the hypothesis that MC1R sequence variation is associated with interspecific variation in unmelanized plumage pattern elements in Old World leaf warblers (genus Phylloscopus). This genus is characterized by a variety of plumage patterns that nonetheless vary along similar lines. Species vary in the presence or absence of pale (unmelanized) pattern elements against a dark background, and these patterns are used in species recognition and courtship. We sequenced most of the MC1R coding region for eight Phylloscopus species, representing the full range of plumage patterns found in this genus. Although MC1R sequence varied among species, this variation was not related to melanin-based plumage variation. Rather, evolution of this locus in these birds appears to be conservative. Ratios of nonsynonymous to synonymous substitutions (dN/dS) were consistently low, suggesting that strong purifying selection has operated at this locus, and likelihood ratio testing revealed no evidence of variable selective pressures among lineages or across codons. Adaptive evolution at MC1R may be constrained by the adaptive importance of plumage pattern elements in this genus.

Animals↗

Stabilizing selection on behavior and morphology masks positive selection on the signal in a salamander pheromone signaling complex.

Natural selection maintains the integration and coordination of sets of phenotypic characters that collectively perform a task. In functional complexes in which characters span molecular to behavioral levels of organization, we might then expect similar modes of selection to produce similar patterns in evolutionary divergence at each level. To test this expectation, we diagnosed selection at behavioral, morphological, and molecular levels for courtship pheromone signaling by plethodontid salamanders. At the levels of morphology and behavior tens of millions of years of stasis (stabilizing selection) occur on each side of a transition from vaccination to olfactory delivery modes. As a proxy for the molecular level, we used plethodontid receptivity factor (PRF), a protein that is an active component of the pheromone. We cloned PRF from 12 Plethodon spp. spanning the delivery transition and obtained multiple alleles from each individual surveyed. Analyses of 61 alleles for PRF identified elevated nonsynonymous over synonymous substitution rates along lineages in a molecular phylogeny, and at 8% of sites in the protein, indicating that positive (directional) selection has acted on this vertebrate pheromone gene. Structural models showed PRF is in a family of cytokines characterized by a four-alpha-helix bundle. Positive selection in PRF was associated with receptor binding sites that are under purifying selection in other cytokines of that family. The evolutionary dynamics of the plethodontid pheromone delivery complex consists of stabilizing selection on morphological and behavioral aspects of signal delivery but positive selection on the signal mediated by receptors. Thus, different selection modes prevail at different levels in this reproductive functional complex. Evolutionary studies of integrated sets of characters therefore require separate analyses of selective action at each level.

Amino Acid Sequence↗

Purification and cDNA cloning of HeLa cell p54nrb, a nuclear protein with two RNA recognition motifs and extensive homology to human splicing factor PSF and Drosophila NONA/BJ6.

While searching for a human homolog of the S.cerevisiae splicing factor PRP18, we found a polypeptide that reacted strongly with antibodies against PRP18. We purified this polypeptide from HeLa cells using a Western blot assay, and named it p54nrb (for nuclear RNA-binding protein, 54 kDa). cDNAs encoding p54nrb were cloned with probes derived from partial sequence of the purified protein. These cDNAs have identical coding sequences but differ as a result of alternative splicing in the 5' untranslated region. The cDNAs encode a 471 aa polypeptide that contains two RNA recognition motifs (RRMs). Human p54nrb has no homology to yeast PRP18, except for a common epitope, but is instead 71% identical to human splicing factor PSF within a 320 aa region that includes both RRMs. In addition, both p54nrb and PSF are rich in Pro and Gln residues outside the main homology region. The Drosophila puff-specific protein BJ6, one of three products encoded by the alternatively spliced no-on-transient A gene (nonA), which is required for normal vision and courtship song, is 42% identical to p54nrb in the same 320 aa region. The striking homology between p54nrb, PSF, and NONA/BJ6 defines a novel phylogenetically conserved protein segment, termed DBHS domain (for Drosophila behavior, human splicing), which may be involved in regulating diverse pathways at the level of pre-mRNA splicing.

Amino Acid Sequence↗

Response to selection for increased hybridization between Drosophila melanogaster females and D. simulans males.

Females of Drosophila melanogaster and males of D. simulans hybridizing in a nonchoice condition were artificially selected for 12 generations. The frequency of hybridization increased from 10% to 79%. Response to selection occurred in both species, particularly in D. melanogaster. Female receptivity was the primary sexual trait that accounted for breaking up sexual isolation in these species, but it remained unclear which elements of the D. simulans male courtship were involved.

Animals↗

Male broiler breeder fowl display high levels of aggression toward females.

Commercial broiler breeder males are reported to display high levels of aggression, often injuring and sometimes killing females. Aggression toward females by mature male domestic fowl is rare and may be regarded as aberrant behavior. The objective of this study was to determine the effects of genetic strain and feed restriction on the sexual and aggressive behavior of male domestic fowl. A 3 x 2 factorial design was used to compare the behavior of three genetic strains (two broiler breeder strains and one commercial laying strain) on two feeding regimens (one restricted and one fed ad libitum) between 25 and 37 d of age. At 22 wk of age, pairs of same-strain males were penned with 20 females of a broiler breeder strain. Behavior was sampled from each pen for six 10-min periods during Weeks 25, 27, 29, 33, and 37. Broiler breeder males displayed significantly more male-to-male (P < 0.0197) and male-to-female aggression (P < 0.0005) than laying strain males. Broiler breeder males chased females (P < 0.0001), forced more copulations (P < 0.0003), and displayed little courtship behavior (P < 0.0001) compared with laying strain males. There were no behavioral differences between broiler breeder strains. It was expected that feed restriction would result in increased aggression. However, males fed ad libitum displayed the most male-to-male (P < 0.0035) and male-to-female (P < 0.0273) aggression. Sexual behavior was not affected by feeding regimen. In conclusion, broiler breeder males display aberrant sexual behavior and extremely high levels of aggression, which are associated with genetic differences, not feed restriction.

Aggression↗

17alpha-ethinylestradiol reduces the competitive reproductive fitness of the male guppy (Poecilia reticulata).

Whether endocrine disruption in an individual male is actually translated into reduced reproductive success in a natural competitive environment is extremely difficult to predict. Here, we have used paternity analysis to provide new information on the ability of an endocrine disruptor to deleteriously affect male guppy reproductive fitness by including the effect of intermale competition. Groups of male guppies were exposed to 10.5, 44.4, or 112 ng/L of the synthetic estrogen 17alpha-ethinylestradiol (EE2) from birth to adulthood. Subsequently, an exposed male competed against an unexposed male for the opportunity to fertilize a receptive female. The successful males siring the majority of the offspring in each brood were then identified using microsatellites in genetic paternity analysis. Only the highest dose of EE2 produced harmful effects with a significantly female-biased sex ratio, significant reductions in male sperm count, testis weight, body coloration and courtship behavior, and a significant increase in body size. These feminizing effects were translated into a highly significant reduction in fertility, where only 1 of the 17 exposed males sired offspring in competition with unexposed males. The evidence suggests that EE2-treated males have reduced reproductive fitness compared with untreated males, possibly the result of EE2 effects on multiple fitness traits. To our knowledge, this is the first study providing evidence of endocrine disruption at the population level that has included the ecologically highly relevant effect of sexual competition on male reproductive fitness.

Animals↗