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Regulation of cell number in Drosophila.

During animal development, different parts grow independently (such as the left and right hands) but they stop growing when they reach the correct size. In most insects, growth of the epidermis is so controlled that, at each moult, there is a precise and proportionate increase in cell number. The mechanisms responsible for this size regulation are not known, but rigid programming of the number of cell divisions is not a requirement as even sister cells in an epithelial sheet divide variably. In the abdomen of dipterans, such as Drosophila, the opportunity for regulation is limited, because mitoses occur only in the embryo and during metamorphosis and not during larval growth. Here we used embryos with a reduced number of cells in the abdominal primordia to determine whether they can regulate towards the normal during subsequent growth. In contrast to expectations, we find no evidence for regulation of cell number.

Animals↗

A hierarchical analysis of the genetic structure of an aquatic insect Bungona (Baetidae: Ephemeroptera).

Recent studies of the genetic structure of stream-dwelling organisms have suggested that fine-scale patterns are the consequence of patchy recruitment from a small number of matings and limited in-stream dispersal. Predictions of this hypothesis were tested by spatial and temporal analysis of the genetic structure of populations of a stream mayfly (Bungona sp: Baetidae) in subtropical streams in south-eastern Queensland. Significant departures from Hardy-Weinberg proportions occurred more often than would be predicted by chance alone and no consistent pattern was observed across sites, loci or sampling times. As in previous studies, the largest differentiation was observed at the smallest spatial scale (reaches within streams) on most sampling occasions. These data provide additional support for a patchy recruitment hypothesis. Despite the fine-scale population structure, there was evidence of widespread adult dispersal across the study region, especially between streams and subcatchments within the same block of continuous dense forest.

Animals↗

Molecular cloning and functional expression of the first insect FMRFamide receptor.

FMRFamide and FMRFamide-related neuropeptides are extremely widespread and abundant in invertebrates and have numerous important functions. Here, we have cloned a Drosophila orphan receptor, and stably expressed it in Chinese hamster ovary cells. Screening of a peptide library revealed that the receptor reacted with high affinity to FMRFamide (EC50, 6 x 10(-9) M). The intrinsic Drosophila FMRFamide peptides are known to be synthesized as a large preprohormone, containing at least 13 related FMRFamide peptides (8 distinct FMRFamides). Screening of these intrinsic Drosophila FMRFamides showed that the receptor had highest affinity to Drosophila FMRFamide-6 (PDNFMRFamide) (EC50, 9 x 10(-10) M), whereas it had a somewhat lower affinity to Drosophila FMRFamide-2 (DPKQDFMRFamide) (EC50, 3 x 10(-9) M) and considerably less affinity to the other Drosophila FMRFamide-related peptides. To our knowledge, this article is the first report on the molecular identification of an invertebrate FMRFamide receptor.

Amino Acid Sequence↗

Molecular cloning of three cDNAs encoding putative larval cuticle protein expressed differentially after larval ecdysis from the mulberry longicorn beetle, Apriona germari.

Three cDNAs encoding putative larval cuticle protein (LCP) were cloned from the mulberry longicorn beetle, Apriona germari. The three cDNA sequences were 309 bp, 396 bp and 408 bp in length, encoding 103, 132 and 136 amino acid residues, respectively. The predicted molecular masses for these LCPs were approximately 9.2 kDa (AgLCP9.2), 12.3 kDa (AgLCP12.3) and 12.6 kDa (AgLCP12.6). Pairwise identity among AgLCP9.2, AgLCP12.3 and AgLCP12.6 were relatively low. Each AgLCP contained a type-specific consensus sequence identifiable in other insect cuticle proteins. The deduced amino acid sequence of AgLCP9.2 is most similar to Bombyx mori LCP18 and those of AgLCP12.3 and AgLCP12.6 are both most similar to B. mori LCP17. Northern blot analysis revealed that the three AgLCPs showed epidermis-specific expression. The expression profile of AgLCPs after larval ecdysis revealed by Northern blot analysis that the high-level mRNA expression of AgLCPs was detected on the first day of larval ecdysis for AgLCP9.2, on the fifth day for AgLCP12.3 and from the first day of larval ecdysis to the fifth day after larval ecdysis for AgLCP12.6, demonstrating that AgLCP mRNAs are differentially expressed in epidermis after larval ecdysis.

Amino Acid Sequence↗

Molecular cloning and structural analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase of the moth Agrotis ipsilon.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which plays a key role in isoprenoid biosynthesis, catalyses the synthesis of mevalonate from HMG-CoA. Insects do not synthesize cholesterol de novo, rather mevalonate derivatives lead to non-sterol isoprenoids which are essential for development and reproduction. In this paper, we describe an HMG-CoA reductase of the moth Agrotis ipsilon and we report its expression in fat body, ovary, muscle, brain and corpora allata tissues of adult specimens. The analysis of the cDNA reveals that it encodes a polypeptide of 833 amino acids (Mr = 89785). Alignments of this HMG-CoA reductase from A. ipsilon with the homologous sequences of other eukaryotes shows a high degree of conservation in all species studied. Parsimony analysis based on these alignments produced dendrograms congruent with the current systematic schemes. This suggests that, during eukaryote evolution, HMG-CoA reductase diversified in parallel with taxonomic splitting.

Amino Acid Sequence↗

Drosophila melanogaster: an insect model for fundamental studies of sleep.

In 2000, Drosophila melanogaster joined the ranks of vertebrates and invertebrates with a defined behavioral sleep state. The characterization of this sleep state revealed striking similarities to sleep in humans: sleep in flies has both circadian and homeostatic components, it is influenced by sex and age, and it is affected by pharmacological agents such as caffeine and antihistamines. As in mammals, arousal thresholds in flies increase with sleep deprivation. Furthermore, changes in brain electrical activity accompany the change from wake to sleep states. Not only do flies and vertebrates share these behavioral and physiological traits of sleep, but they are likely to share at least some genetic mechanisms underlying the regulation of sleep as well. This article reviews the methods currently used to identify and characterize the Drosophila sleep state. As these methods become more refined and our understanding of Drosophila sleep more detailed, the powerful techniques afforded by this organism are likely to unveil deep insights into the function(s) and regulatory mechanisms of sleep.

Animals↗

Transcriptional regulation of an unusual trypsin-related protein expressed during insect metamorphosis.

The full-length cDNA for a trypsin-related protein expressed during larval-pupal metamorphosis was obtained. The encoded N-terminal half of the protein possessed similarity to trypsin proteases, including the correctly positioned histidine and aspartic acid members of the catalytic triad. However, the remainder of the encoded protein bore little resemblance to trypsin, and was altogether missing the canonical sequence containing the catalytic serine residue. The product of in vitro transcription/translation of the cDNA was a 28 kDa protein. Under normal conditions of a declining juvenile hormone titer, transcription of the mRNA as a proportion of total genomic transcription steadily increased for the first 3 days of the final stadium, but this increase was delayed and suppressed by maintenance of a high juvenile hormone titer. During the normal increase in transcription on day 3 of the final stadium, a sharp decline was observed in the steady-state abundance of the transcript, measured relative to abundance of the remaining mRNAs, suggesting that the stability of the message decreases after tissue commitment for a pupal molt.

Amino Acid Sequence↗

Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures.

Rabies virus nucleoprotein (N) was produced in insect cells using the baculovirus expression system described by Préhaud et al. (Virology 178, 486-497, 1990). The protein was either purified on a CsCl gradient, resulting in a mixture of nucleocapsid-like structures and beaded rings, as observed by electron microscopy, or on a glycerol gradient that resulted in a preparation of the rings only. The rings and nucleocapsid-like structures had the same morphological characteristics as viral nucleocapsids. N in these structures is an 84 A long and thin molecule that is spaced at around 34 A along the length of the nucleocapsid, identical in shape and spacing as the nucleoprotein in nucleocapsids of rabies virus and very similar to those of vesicular stomatitis virus. The recombinant nucleocapsids contained RNA with a stoichiometry similar to that found in viral nucleocapsids. The RNA bound in the beaded rings was a subset of the insect cellular RNA. One of the RNA species was partially sequenced and, although a positive identification could not be made, could correspond to a tRNA. With respect to sensitivity to trypsin and RNase digestion, the recombinant and viral nucleocapsids behaved similar. Trypsin cleaved a 17 kDa fragment from the carboxy terminus of N with only a very small effect on the morphology of the nucleocapsids. RNase A completely digested the resident RNA in both viral and recombinant nucleocapsids into fragments of 4-5 nt long, again with no effect on the morphology of the nucleocapsids. Thus, when the RNA is cleaved, the structure must be maintained by protein-protein contacts. Experiments to remove the resident RNA from viral and recombinant rabies virus nucleocapsids failed, whereas the same methods used to eliminate the RNA from vesicular stomatitis virus nucleocapsids was successful.

Animals↗

Modeling the structure of the type I peritrophic matrix: characterization of a Mamestra configurata intestinal mucin and a novel peritrophin containing 19 chitin binding domains.

Twelve to fourteen integral proteins were found to reside in the Type I peritrophic matrix (PM) of Mamestra configurata (bertha armyworm) larvae. Several methods were employed, including de novo peptide sequencing, the generation of a midgut-specific EST database and immunological screening, which led to the isolation of cDNAs encoding two integral PM proteins. McPM1, the largest PM protein described to date at 202 kDa, was comprised of a concatamer of 19 chitin binding domains (CBD), 12 of which resided within a central repetitive region consisting of six iterations of a two CBD module. The protein was found to reside within the PM primarily as several lower molecular weight, presumably proteolytically processed, forms. McMUC1 was similar in structure to other insect intestinal mucins (IIM) and was highly glycosylated. The expression of both proteins was restricted to the larval midgut. Lower molecular weight proteins that may represent non- and partially glycosylated forms of McMUC1 were also recognized by an anti-McMUC1 antiserum. These were preferentially degraded upon ingestion of M. configurata multi-capsid nucleopolyhedrovirus by larvae, possibly by a viral-encoded metalloprotease. A molecular model of PM structure is presented featuring the interaction of McPM1 with chitin inter-fibril junctions and McMUC1 with the extended chains in the internodal regions. The potential for interaction between PM proteins via intermolecular disulfide bond formation and through association of CBD with N-linked glycans is discussed.

Amino Acid Sequence↗

Quantification of juvenile hormone III, vitellogenin, and vitellogenin-mRNA during the oviposition cycle of the lubber grasshopper.

The vitellogenic cycle of the lubber grasshopper (Romalea microptera) was studied by measuring levels of juvenile hormone (JH III), vitellogenin, and vitellogenin-mRNA through the first oviposition cycle. JH III and vitellogenin were measured by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), respectively. To measure vitellogenin-mRNA, a partial (753 bp) cDNA fragment of vitellogenin was isolated from the fat body of vitellogenic animals. The sequence of this cDNA was related to vitellogenin sequences in other insect species. Using these sequence data, an RT-PCR (reverse transcriptase polymerase chain reaction) assay was developed to quantify vitellogenin-mRNA levels during the oviposition cycle. Vitellogenin-mRNA levels in the fat body tissue from virgin females were measured on specific days after eclosion and compared to hemolymph levels of JH III and vitellogenin from the same individuals. The levels of all three compounds (JH III, vitellogenin, and vitellogenin-mRNA) showed similar changes throughout the oviposition cycle, being undetectable or nearly undetectable initially (day 3), rising to maximum levels on days 23 and 28, and then dropped to lower or undetectable levels on the day of oviposition. The ability to measure these characteristics will be useful for studying the effects of hormonal and nutritional manipulations on reproduction.

Animals↗

Differentiation of Asian rice gall midge, Orseolia oryzae (Wood-Mason), biotypes by sequence characterized amplified regions (SCARs).

We developed a polymerase chain reaction (PCR)-based assay that distinguished five different biotypes of the Asian gall midge (Orseolia oryzae), a major insect pest of rice. A total of 400 random primers were screened using random amplified polymorphic DNAs (RAPDs). Five diagnostic PCR products were isolated, cloned, sequenced and converted to sequence characterized amplified regions (SCARs). Primers specific to these SCARs were able to amplify specific DNA fragments from genomic DNAs of five biotypes of gall midge in a multiplexed-PCR-based assay. The amplified DNA fragments were used as diagnostic markers to identify different biotypes of gall midge. The SCAR primers were also capable of differentiating the Asian from the African rice gall midge (Orseolia oryzivora) as well as detecting a variant of biotype 5 which caused an outbreak in Kerala, India. Unlike the use of plant host differentials and midge feeding behaviour for identifying biotypes, this assay is fast, reliable and unaffected by environmental factors.

Animals↗

A phylogenetic analysis of soldier evolution in the aphid family Hormaphididae.

Aphid soldiers, altruistic larvae that protect the colony from predators, are an example of highly social behaviour in an insect group with a natural history different from the eusocial Hymenoptera and Isoptera. Aphids therefore allow independent tests of theory developed to explain the evolution of eusociality. Although soldiers have been discovered in five tribes from two families, the number and pattern of origins and losses of soldiers is unknown due to a lack of phylogenetic data. Here I present a mtDNA based phylogeny for the Hormaphididae, and test the hypothesis that soldiers in the tribe Cerataphidini produced during two points in the life cycle represent independent origins. The results support this hypothesis. In addition, a minimum of five evolutionary events, either four origins and one loss or five origins, are required to explain the distribution of soldiers in the family. The positions of the origins and losses are well resolved, and this phylogeny provides an historical framework for studies on the causes of soldier aphid evolution.

Animals↗

Identification of a cyclic nucleotide- and voltage-activated ion channel from insect antennae.

From an antennal cDNA library of Heliothis virescens a clone has been isolated encoding a polypeptide of 678 amino acids. Data base comparisons and primary structure analysis of the deduced protein sequence (HvCNG) indicated significant homology to cyclic nucleotide and voltage-activated ion channels including six putative membrane spanning domains, a putative cyclic nucleotide binding site, a pore region and a voltage-sensor motif. Heterologous expression of the cloned cDNA in Sf9 cells resulted in a polypeptide of the predicted molecular mass. Patch clamp analysis allowed to record the activity of the identified HvCNG channels; they were activated by cAMP but also by hyperpolarization. The channel displayed in potassium solution a conductance of 30 pS; the ion selectivity was calculated as PK/PNa approximately 3. Northern blot analysis revealed that the channel is highly expressed in the antennae; weaker signal were detected in heads and legs. In situ hybridization of tissue sections through the antennae showed a spatial distribution of reactive cells; they are located beneath sensillar hairs. Thus, a novel channel type has been identified which may play an important role in antennal cells.

Amino Acid Sequence↗

Repeated losses of TTAGG telomere repeats in evolution of beetles (Coleoptera).

We studied the occurrence of (TTAGG)n telomere repeats in 12 species of beetles, representing main lineages of the Coleoptera phylogenetic tree, by Southern hybridization and fluorescence in situ hybridization (FISH). In contrast to other insect orders, beetles were heterogeneous with respect to the occurrence of TTAGG repeats. In addition, the presence or absence of (TTAGG)n motif was irrespective of phylogenetic relationships. In the suborder Polyphaga, six species displayed positive hybridization signals. These were Silpha obscura, Agrilus viridis, Ampedus sanguineus, Stegobium paniceum, Oryzaephilus surinamensis, and Leptinotarsa decemlineata. Whereas negative signals were obtained in three polyphagan species, Geotrupes stercorarius, Thanasimusformicarius, and Sitophilus granarius. In the suborder Adephaga, the TTAGG sequence was present in one species, Graphoderus cinereus, and absent in two species, Orectochilus villosus and Pterostichus oblongopunctatus. We concluded that the telomerase-dependent (TTAGG)n motif had been repeatedly lost in different phylogenetic branches of Coleoptera and probably replaced with another mechanism of telomere elongation. This had to happen at least 5-6 times. The results suggest a predisposition or a backup mechanism of telomere maintenance in the genome of beetles that enabled them to make frequent evolutionary changes in the telomere composition.

Animals↗

Targeted ablation of glia disrupts axon tract formation in the Drosophila CNS.

Glial cells are thought to play a role in growth cone guidance, both in insects and in vertebrates. In the developing central nervous system of the Drosophila embryo, the interface glia form a scaffold prior to the extension of the first pioneer growth cones. Growing axons appear to contact the glial scaffold as the axon tracts are established. We have used a novel technique for targeted cell ablation to kill the interface glia and thus to test their role in establishment of the embryonic axon tracts. We show that ablation of the interface glia early in development leads to a complete loss of the longitudinal axon tracts. Ablation of the glia later in embryonic development results in defects comprising weakening and loss of axon fascicles within the connectives. We conclude that the interface glia are required first for growth cone guidance in the formation of the longitudinal axon tracts in the Drosophila embryo and then either to direct the follower growth cones, or to maintain the longitudinal axon tracts.

Animals↗

Sequence and evolution of the gene for the monomeric globin I and its linkage to genes coding for dimeric globins in the insect Chironomus thummi.

We isolated genomic clones containing sequences encoding globins I and IA from a Chironomus thummi thummi genomic library. Three clones contain globin IA (ctt-1A) genes, while one contains a globin I (ctt-1) gene. The coding regions of the four genes are identical except for the single base substitution accounting for the globin I/IA polymorphism. The noncoding DNA flanking the coding region is more than 98% similar, confirming a previous hypothesis that the globin ctt-1 and ctt-1A genes are alleles. Hemoglobins I and IA are monomeric in the insect hemolymph. Earlier in situ hybridization studies suggested that monomeric and dimeric globin genes are clustered at different chromosomal loci. In situ hybridization of ctt-1 DNA to polytene salivary gland chromosomes places the ctt-1 gene on the same band as genes for the dimeric globins II beta and VIIB, forcing revision of the earlier hypothesis that genes for monomeric and dimeric globin genes are at different loci. The evolution of the ctt-1 and ctt-1A alleles and of the two globin gene loci are discussed.

Amino Acid Sequence↗

Requirements of Kettin, a giant muscle protein highly conserved in overall structure in evolution, for normal muscle function, viability, and flight activity of Drosophila.

Kettin is a giant muscle protein originally identified in insect flight muscle Z-discs. Here, we determined the entire nucleotide sequence of Drosophila melanogaster kettin, deduced the amino acid sequence of its protein product (540 kD) along with that of the Caenorhabditis elegans counterpart, and found that the overall primary structure of Kettin has been highly conserved in evolution. The main body of Drosophila Kettin consists of 35 immunoglobulin C2 domains separated by spacers. The central two thirds of spacers are constant in length and share in common two conserved motifs, putative actin binding sites. Neither fibronectin type III nor kinase domains were found. Kettin is present at the Z-disc in several muscle types. Genetic analysis showed that kettin is essential for the formation and maintenance of normal sarcomere structure of muscles and muscle tendons. Accordingly, embryos lacking kettin activity cannot hatch nor can adult flies heterozygous for the kettin mutation fly.

Amino Acid Sequence↗

F2 screen variations and associated statistics.

The F2 screen is a flexible methodology used to estimate the frequency of resistance alleles (R) in an insect population. We have developed several alternative protocols, along with the associated statistics, to conduct an F2 screen with mated or unmated individuals, random and nonrandom mating of F1 adults, and the screening of multiple lines together in the F2 screen. Our protocols describe how to perform and analyze an F2 screen starting with unmated P1 as an alternative to mated females. A randomly mated population of > or = 50 F1 adults should be sufficient to detect R alleles > 99% of the time. If nonrandom mating occurs in the F2 screen, it is most likely to be positive assortative mating, and this would improve the probability of detecting an R allele. Pair mating the F1 adults greatly increases costs of the screen while providing a small, but negligible improvement in detecting R alleles. The number of screens may be reduced by more than two-thirds by screening multiple lines together. These methodological variants show the F2 screen to be much more robust than originally described.

Animals↗