Regulatory elements involved in the tissue-specific expression of the yellow gene of Drosophila

Summary We have assessed the DNA sequence requirements for the correct spatial pattern and phenotypic expression of y in the late embryo/larvae. The wild-type larval phenotype requires both the regions between-294 bp and-92 bp and a portion of the intron; the sequence element(s) located within the intron can act in a position independent manner to effect the wild-type larval phenotype. The larval expression pattern was examined by tissue experiments in situ and by staining germline transformants derived from various y/lacZ fusion constructs. The larval expression of y is restricted to the mouthparts, microsetae and anal plates. While the-495 bp to+194 bp region alone cannot effect a wild-type larval expression pattern, this region in conjunction with the intron appears to be sufficient to drive -gal expression in an essentially wild-type pattern. Our data further suggest that the-294 bp to-92 bp region contains elements which specify the larval pattern and that the element(s) in the intron normally act to enhance the level of expression necessary for the wild-type larval phenotype. We also present a phenotypic analysis of the adult cuticle structures of germline transformants derived from a variety of deletion and rearrangement constructs of the y gene. This analysis has revealed several new features associated with the regulation of y expression.     

A mutation affecting larval muscle development in Drosophila melanogaster

The phenotypic analysis of a new spontaneous recessive lethal mutation of Drosophila melanogaster is described. The lethal(2)thin mutation maps at 85.6 on chromosome 2 and produces a characteristic long, thin puparium due to an inability to shorten the larval form prior to pupariation. Histological examination of larval muscles and behavioural studies support the hypothesis that the mutation affects the striated structure of the larval muscles in late larval stages. Lethality largely occurs due to an inability to perform the movements necessary for pupation, although there is evidence for larval and possibly embryonic lethal phases.     

Pupal polymorphism in the butterfly Danaus chrysippus (L.): environmental, seasonal and genetic influences

The pupae of the tropical butterfly Danaus chrysippus are either green or pink the switch being operated by a ‘greening’ hormone produced in the larval head. Both environmental and genetic cues are involved in controlling the endocrine mechanism. The environmental factors identified are of two distinct kinds: proximate factors influence pupal colour after the larva has selected its pupation site, whereas ultimate factors are effective at an earlier stage, either prompting choice of pupation site by the larva or priming pupation physiology in a particular direction. Genetic factors preadapt the larva to form a pupa which will be cryptic in the normal or average conditions, climatic or biogeographical, anticipated in its environment. The proximate factors demonstrated are background colour, darkness, light quality (wavelength) and humidity. There is some evidence that substrate texture may also be relevant. Ultimate factors are temperature, humidity and species of larval foodplant. Two closely linked gene loci which govern the phenotype of adult morphs and races either have a pleiotropic effect on pupa colour or are closely linked with other genes which do so. Moreover, the two loci interact epistatically with respect to their pupation effects. Factors producing predominantly green pupae are plant substrates, yellow background, darkness, yellow light, high humidity, high temperature, the b allele at the B locus when homozygous and, on non-plant substrates, the C allele at the C locus. High frequencies of pink pupae result on non-plant substrates, red backgrounds, in blue light, low humidity, low temperatures and in B- and cc genotypes. The C locus alleles, C and c, interact epistatically with the B alleles, their effect on choice of pupation site being determined by linkage phase. Of the two foodplants tested, Calotropis produced a high frequency of green pupae and Tylophora of pinks. The seasonal cycling of rainfall, temperature, availability or condition of foodplant, and gene frequencies are all correlated with oscillations in the frequencies of green and pink pupae. Though genotype influences pupa colour, all genotypes are capable of forming pupae of both colours. The variation can therefore be attributed to an environmental polyphenism superimposed upon a genetic polymorphism. The hormone producing green pupae emanates from the head during the prepupal period. Denied hormonal influence, the pupa is pink. Pupal colour is judged to be aposematic at close range and cryptic at distance.     

A description of larval and early juvenile development in Paralomis spinosissima (Decapoda: Anomura: Paguroidea: Lithodidae) from South Georgia waters (Southern Ocean)

The early ontogenetic stages of Paralomis spinosissima Birstein and Vinogradow, 1972, are described in detail and illustrated, with notes on morphological variability observed. Larval and early juvenile development was described to the crab I instar reared under controlled conditions of temperature and food supply. The abbreviated larval development invariably passed through two zoeal stages and the benthic megalopa stage. The larval development was completed without food supply, and food Artemia nauplii were first given after moult to the crab-I stage. Simplification and retarded development of the mouthparts are discussed as a function of lecithotrophy of these larvae and based on morphology no facultative feeding mode is suggested. Lecithotrophy in the Southern Ocean Lithodidae is discussed to be an adaptation allowing independence from seasonal food availability at high latitudes.     

An unusual barrier to gene flow: perpetually immature larvae from inter-population crosses in the flour beetle, Tribolium castaneum

We genetically characterize an unusual hybrid incompatibility phenotype manifest in F₁ offspring of crosses between two populations of Tribolium castaneum. Hybrid larvae cease development at the third larval instar, persisting as ‘perpetually immature larvae’ thereafter. Although unable to produce viable adult hybrid offspring with one another, each population produces abundant, fertile hybrids with other populations, indicating a recent origin of the incompatibility and facilitating genetic studies. We mapped the paternal component of the hybrid phenotype to a single region, which exhibits two characteristics common to hybrid incompatibility: marker transmission ratio distortion within crosses and elevated genetic divergence between populations. The incompatible variation and an elevation in between‐population genetic divergence is associated with a region containing the T. castaneum ecdysone receptor homologue, a major regulatory switch, controlling larval moults, pupation and metamorphosis. This contributes to understanding the genetics of speciation in the Coleoptera, one of the most speciose of all arthropod taxa.     

Phylogenetic analysis of Myxophaga (Coleoptera) using larval characters

A phylogenetic parsimony analysis of fifty-four larval characters of Myxophaga (excluding Lepiceridae) resulted in two minimal length cladograms. The monophyly of Torridincolidae, Hydroscaphidae and Microsporidae is supported by several autapomorphies: miniaturization, flattened body with laterally extended tergites, broadened head, scale-like surface structures, broad tentorial bridge, disc-shaped labral sensilla, spiracular gills and pupation in the last larval exuviae. Hydroscaphidae are the sister group of Microsporidae. Larvae of both families are characterized by semi-entognathous mouthparts, tergites with posterior rows of lancet-shaped setae, claws with flattened basal spines and balloon-shaped spiracular gills. The monophyly of all families is supported by autapomorphies. Torridincolidae excluding Delevea is defined as a monophylum by four derived character states: body ovoid, thorax semicircular and as long as abdomen, labral sensilla fused and abdominal sternite IX distinctly reduced and triangular. The monophyly of Torridincolinae (sensu Endrödy-Younga 1997b) is supported by two autapomorphies. The proposed branching pattern suggests that the early representatives of Myxophaga (excluding Lepiceridae) were living in aquatic conditions with a preference for hygropetric habitats. The tendency to live on rocks in running water and miniaturization have played an important role in myxophagan evolution.     

The biology of the black larval mutant of the tobacco hornworm, Manduca sexta

A mutant of the tobacco hornworm, Manduca sexta, was found to form black melanized cuticle in the last larval instar. This black phenotype is due to a single sex-linked gene whose expression can be changed by one or more modifier genes. The expression of the mutant phenotype is prevented by juvenile hormone (JH) application at the time of head cap apolysis during the moulting cycle to the last larval instar. The bl mutant is equally as sensitive to JH at this time as is a neck-ligated wild type larva, ruling out an absence of hormone receptors or a difference in JH metabolism. The bl corpora allata were found to be less active at this time than were those of the wild type larva, suggesting that the defect resides in the control of the corpora allata. Since selection for complete expression of the bl phenotype is easy, this mutant provides the basis for an ultrasensitive JH bioassay to be described in a forthcoming paper.     

Mouthpart and foregut ontogeny in larval, postlarval, and juvenile spiny lobster, Panulirus argus Latreille (Decapoda, Palinuridae)

The spiny lobster Panulirus argus has a life cycle consisting of a long-term (～9-12 months) planktonic larval period with 11 larval stages (the phyllosoma), a short (<1 month?) planktonic-to-benthic transitional postlarval stage (the puerulus), and benthic juvenile and adult phases. The mouthparts and foregut during these stages were examined and described by means of scanning electron microscopy (SEM) in an investigation of the species' developmental morphology, diet, and ecology. The phyllosoma mouthparts close to the esophagus are the labrum, mandibles, paragnaths, and first maxillae. The second maxillae and first and second maxillipeds are increasingly distant from the esophagus as the larva develops. The pair of asymmetrical mandibles bear many teeth and spines, and the molar processes form what appears to be an intricate toothed shear. The mandibles remain similar throughout the phyllosoma stages. During the molt into the puerulus, the mouthparts are greatly changed, and the second maxilla and the three maxillipeds join the other mouthparts near the esophagus. However, the transformation appears incomplete, and many of the mouthparts are not fully formed until the molt to juvenile completes their development. The phyllosoma foregut lacks a gastric mill and has but one chamber. In addition, the first two stages lack a gland filter. During the molt to puerulus, the foregut is greatly changed and subsequently is similar to typical decapod foreguts in having an anterior cardiac and posterior pyloric chamber. Only rudimentary internal armature is present. Following the molt to juvenile, the foregut is quite similar to that of the adult, which exhibits a substantial gastric mill. The 11 phyllosoma stages were separated into two groups (group A = stages 1-5, group B = stages 6-11) on the basis of changes in both mouthpart and foregut morphology. The puerulus has never been observed to feed. Nothing was observed in our investigations that would prevent feeding, though both mouthpart and foregut development appeared incomplete. The mouthpart and foregut structures of larval, postlarval and juvenile P. argus differ widely, possibly reflecting the extreme modifications for different habitats found among these life phases.     

Neural development in an imaginal disc mutant of Drosophila melanogaster

The neural phenotype of an imaginal disc degenerate mutant l(1)d deg-3 was studied in histological sections. The mutant larvae showed severe abnormalities in the imaginal neural development. Gynandromorphs, which are composed of genetically mutant and nonmutant cells, were generated and analyzed as late larvae. The results of mosaic analysis were consistent with l(1)d deg-3 gene acting autonomously in the imaginal disc and imaginal neural cells. The optic lobe development patterns observed in the larval mosaics provided evidence for an eye disc-optic lobe interaction during the late third instar larval stage.     

Rheoptilum: A New Genus of Two-Tailed Baetidae (Ephemeroptera) from Madagascar

Rheoptilum gen. n. is established for R. arni sp. n. and R. lokohensis sp. n. from Madagascar. Imaginal and larval stages are both described. Rheoptilum nymphs have two important adaptations: mouthparts are highly modified for scraping and the habitus is adapted for fast flowing waters. The genus possesses an extremely broad labrum with distal margin almost straight, incisors almost fused, right prostheca long and slender, caudal filament reduced to a single segment, legs long and slender, dorsally bordered with long and thin setae and the body dorsoventrally flattened. This combination of features distinguishes Rheoptilum from other Afro-Malagasy baetid genera. The phylogenetic position of this new genus is discussed.     

Circannual pupation rhythm in the varied carpet beetle Anthrenus verbasci under different nutrient conditions

Anthrenus verbasci pupates in spring and the timing of pupation is controlled by a circannual rhythm. Although A. verbasci is considered to be a univoltine species in Japan, it is assumed that larval development in its natural habitats, including bird nests, varies with nutrient availability, and that the life cycle often takes two or more years to complete. In the present study, larval development and pupation times were compared under constant and outdoor conditions in larvae provided a diet of either high‐nutrient bonito powder or low‐nutrient pigeon feathers. Although a circannual pupation rhythm was observed irrespective of the diet used, larval development was slower on feathers than on bonito powder. The pupation times on feathers varied over three years or more under both constant and outdoor conditions. Under outdoor conditions, larvae grown on feathers needed three years to approach the weight gained within a year by larvae grown on bonito powder. It is considered that life cycle length in A. verbasci is often two years or more in nutritionally unstable natural habitats, and that this species has probably evolved a circannual rhythm as a seasonal adaptation to nutrient‐poor environments.     

Food availability controls the onset of metamorphosis in the dung beetle Onthophagus taurus (Coleoptera: Scarabaeidae)

In nature, larvae of the dung beetle Onthophagus taurus (Schreber 1759) are confronted with significant variation in the availability of food without the option of locating new resources. Here we explore how variation in feeding conditions during the final larval instar affects larval growth and the timing of pupation. We found that larvae respond to food deprivation with a reduction in the length of the instar and premature pupation, leading to the early eclosion of a small adult. To achieve pupation, larvae required access to food for at least the first 5 days of the final instar (= 30% of mean third‐instar duration in control individuals), and had to exceed a weight of 0.08 g (= 58% of mean peak weight in control individuals). Larvae that were allowed to feed longer exhibited higher pupation success, but increased larval weight at the time of food deprivation did not result in increased pupation success except for larvae weighing > 0.14 g. Larvae responded to food deprivation by initiating and undergoing the same sequence of developmental events, requiring the same amount of time, as ad libitum‐fed larvae once those had reached their natural peak weight. Our results reveal a striking degree of flexibility in the dynamics and timing of larval development in O. taurus. They also suggest that premature exhaustion of a larva's food supply can serve as a cue for the initiation of metamorphosis. Premature metamorphosis in response to food deprivation has been documented in amphibians, but this is, to the best of our knowledge, the first time such a behaviour has been documented for a holometabolous insect. We discuss our findings in the context of the natural history and behavioural ecology of onthophagine beetles.     

Salivary gland development in the blowfly,Calliphora erythrocephala

The salivary gland of adult Calliphora erythrocephala is a tubular structure composed of secretory, reabsorptive, and duct regions. Development of these structures has been followed during the six days of larval and ten days of pupal growth. Two small groups of imaginal cells located at the junction between larval gland and duct give rise to the adult gland. These presumptive adult cells divide during all larval stages and appear to be functional components of the larval gland. Shortly after pupation, the larval gland breaks down and the imaginal cells proliferate rapidly, forming sequentially the duct, reabsorptive and secretory regions. Proliferating regions of the developing gland are frequently encrusted with haemocytes. As it elongates the gland establishes intimate contacts first with the basement membrane of the degenerating larval gland, later with an epithelial layer surrounding the main dorsal tracheal trunks, and then with the gut. Cell division continues until about five days after pupation, bu t the gland is unable to secrete fluid in response to 5-hydroxytryptamine stimulation until two hours after the adult fly emerges. The Golgi complex appears to be involved in forming the highly folded membranes of the canaliculi in the secretory region. Presumptive adult salivary gland cells appear to increase in number logarithmically from the time of hatching of the larva until five days after pupation. This contrasts with the development of classical imaginal discs, in which cell division ceases prior to pupation.     

Identification of a genetic network for an ecologically relevant behavioural phenotype in Drosophila melanogaster

Pupation site choice of Drosophila third‐instar larvae is critical for the survival of individuals, as pupae are exposed to various biotic and abiotic dangers while immobilized during the 3–4 days of metamorphosis. This singular behavioural choice is sensitive to both environmental and genetic factors. Here, we developed a high‐throughput phenotyping approach to assay the variation in pupation height in Drosophila melanogaster, while controlling for possibly confounding factors. We find substantial variation of mean pupation height among sampled natural stocks and we show that the Drosophila Genetic Reference Panel (DGRP) reflects this variation. Using the DGRP stocks for genome‐wide association (GWA) mapping, 16 loci involved in determining pupation height could be resolved. The candidate genes in these loci are enriched for high expression in the larval central nervous system. A genetic network could be constructed from the candidate loci, which places scribble (scrib) at the centre, plus other genes known to be involved in nervous system development, such as Epidermal growth factor receptor (Egfr) and p53. Using gene disruption lines, we could functionally validate several of the initially identified loci, as well as additional loci predicted from network analysis. Our study shows that the combination of high‐throughput phenotyping with a genetic analysis of variation captured from the wild can be used to approach the genetic dissection of an environmentally relevant behavioural phenotype.     

Genetic variation in pupation height in a population of Drosophila simulans

Preadult mortality of Drosophila simulans in the laboratory is influenced by larval behavior during pupation site choice. The possibility that larvae select a suitable place to pupate may have profound repercussions on their darwinian fitnesses. Twenty-three isofemale lines of a population of D. simulans were analyzed twice for pupation preference, the first time inmediately after being captured, and again after a year of laboratory culture. Pupation height per vial, pupation height on the vial walls, percentage of pupae either in food or on the wall, and egg-to-pupa viability were estimated. A great intrapopulational variation was noticed with an important genetic component for pupation site choice. This suggests a great larval adaptative potential in the population for selecting and using heterogeneous habitats. Time seems not to have modified the population in both the mean values and the phenotypic and genetic variances. Larval viability, however, increased with time. Pupation site choice is discussed on the basis of two independent sets of genes.     

Nosema takapauensis n.sp., a microsporidan parasite of larvae of Costelytra zealandica (Coleoptera: Scarabaeidae) in New Zealand

Morphological and biological features of a microsporidan protozoan parasite of larvae of Costelytra zealandica collected at Takapau, southern Hawkes Bay, are described and evaluated taxonomically. The parasite multiplies in the fat body of all larval instars, causing massive tissue disintegration in advanced infection resulting in the retardation of development and ultimately death before pupation. The microsporidan forms one spore per sporont, and therefore belongs to genus Nosema’, it is considered to be specifically distinct from its nearest congener, N. melolonthae.     

Development time and pupation behavior in theDrosophila melanogaster subgroup (Diptera: Drosophilidae)

This study is an in-depth analysis of intersexual, intraspecific, and interspecific variability in larvopupal developmental time, pupation site preference, and larval and pupal survival of a number of isofemale lines of the speciesDrosophila mauritiana, D. melanogaster, D. sechellia, D. simulans, D. teissieri, andD. yakuba. There was no significant sex differences in pupation height, but females eclosed significantly earlier than males in all species. In addition, the suggestion of a strong negative correlation between larval developmental time and pupation height could not be confirmed in this study. The hypothesis that differences in pupation height provide a basis for niche partitioning between closely related species with overlapping distributions was tested by three planned orthogonal contrast analyses of variance. First, the two speciesD. teissieri andD. yakuba, with largely overlapping distribution, were significantly different in pupation height. Second, the two allopatric, nonoverlapping island speciesD. mauritiana andD. sechellia did not significantly differ in pupation height. However, the absence of a significant difference in the final contrast between the two cosmopolitan speciesD. melanogaster andD. simulans, which are often found together, makes us cautious to accept the hypothesis.     

Chitin biosynthesis during pupal development of Drosophila melanogaster and the effect of the lethalcryptocephal mutation

Estimation of chitin deposition in the pupal and adult cuticles of adult Drosophila melanogaster during the pupal period is described. The timing of the periods of chitin deposition is compared with that deduced by previous workers using electron microscopy. The hypothesis that lethalcryptocephal mutant homozygotes are unable to evert their cephalic complexes at pupation because of excess chitin deposition is examined. The data obtained show no evidence that the mutation has any effect on chitin deposition.     

Genetics and ecology of Drosophila melanogaster larval foraging and pupation behaviour

In this paper we show that, (1) Drosophila melanogaster larvae utilize a variety of pupal microhabitats in an orchard, (2) variation in larval foraging path length, pupation distance from the food and pupal microhabitat preference (on or off the fruit) is genetically based and, (3) variation in these behaviours can be maintained in a spatially heterogenous environment since there is a reversal in pupation site suitability in wet and dry pupal microhabitats. Differences in path length in both laboratory and natural populations can be attributed to genes on the second pair of chromosomes and is under simple genetic control, whereas differences in pupal height are polygenically inherited (the second pair of chromosomes influences pupal height three times more than the third pair). Pupae collected from on-fruit sites had shorter foraging path lengths and lower pupal heights than off-fruit populations. Populations from the orchard maintained their field pupal microhabitat preferences even after 1 year of rearing them in the laboratory. Larvae with the sitter larval phenotype (short path lengths and low pupal heights tended to pupate more on-fruit than those with the rover phenotype (long path lengths and high pupal heights). To determined if these genetically based differences in microhabitat preference contributed to fitness, larval pupation behaviour was studied in a “field assay” (dish with fruit on soil) with soil water content varied. At low soil water contents, pupal survivorship was significantly better on the fruit whereas, at high soil water contents, survivorship was better in the soil. There was a reversal in which microhabitat (dry or wet) was a better site for pupation. In the field environment where soil water content fluctuates in space and time, such a reversal would explain the maintenance of genetic variation for these larval behaviours. Another selective agent acting on D. melanogaster larvae in our orchard is parasitization by Asobara tabida. This parasitoid parasitizes larvae with high locomotory scores (e.g. rovers) significantly more than those with low scores (sitters). This study relates laboratory phenotypes to field phenotypes thereby linking the ecological, behavioural and genetic components of larval habitat selection in D. melanogaster.     

Genetic control ofHylemya antiqua III. Differences in pupation ability between two strains

Some relevant traits of a wild (L) and a laboratory (C) strain of Hylemya antiqua (Meigen ), determining differences in their pupation ability under experimental conditions have been investigated in relation to genetic control. The wild strain showed an intrinsic higher pupation ability than the laboratory strain. The minimum feeding period was 0.6 days longer for the C strain. The minimum larval dry weight was different for the two strains. With a normal feeding period C larvae pupated on average 1.63 days later than L larvae. By the shortening of the larval feeding period an acceleration of the larval development of both strains was observed: the acceleration of development was more marked for the wild strain. This result has been contrasted with published works on Drosphila. The consequences of these differences as far as the competitive ability of each strain is concerned, have been discussed in relation to genetic control.