Genomic innovations, transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa pest species.

BACKGROUND: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests. RESULTS: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes. CONCLUSIONS: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant.

Txp40, a ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria.

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.

Regulation of lepidopteran foregut movement by allatostatins and allatotropin from the frontal ganglion.

The frontal ganglion and associated neuronal pathways in larvae of the noctuid moth Helicoverpa armigera have been studied immunocytochemically with antisera against the endogenous neuropeptides, the allatostatins (helicostatins), and allatotropin. Two pairs of large ganglionic neurones contain allatostatin immunoreactivity, with the anteriormost of these pairs showing colocalisation with allatotropin. Allatostatin and allatotropin axons exit the frontal ganglion in the recurrent nerve and traverse the surface of the crop to give terminal arborisations around the stomodeal valve. There is a greater degree of lateral branching of allatotropin axons compared with allatostatin axons over the crop musculature. In vitro experiments show that the two types of peptides have antagonistic effects on the spontaneous myoactivity of the crop musculature. Allatotropin is myostimulatory at concentrations as low as 10(-16) M, enhancing both frequency and amplitude of peristaltic waves of contraction. All members of the helicostatin family inhibit peristalsis completely at concentrations of 10(-7)-10(-6) M and, to varying degrees, at 10(-10)-10(-8) M. On the basis of this evidence, it is suggested that peptidergic neurones of the frontal ganglion play a major part in regulating foregut motility through the antagonistic actions of the allatostatins and allatotropin.

Distribution of sulfakinin-like peptides in the central and sympathetic nervous system of the American cockroach, Periplaneta americana (L.) and the field cricket, Teleogryllus commodus (Walker).

We describe the distribution of sulfakinin-like neuropeptides in the central and sympathetic nervous system of the American cockroach Periplaneta americana (L.) (Blattodea) and the field cricket Teleogryllus commodus (Walker) (Othoptera), using an antisulfakinin primary antibody and confocal laser scanning microscopy. We conclude that, in the cockroach, sulfakinin-like material is produced in ten pairs of anterior cells in the pars intercerebralis, as well as two pairs of medial and one major pair of lateral posterior brain cells. This contrasts with findings in other insects, including the cricket, where only the posterior cell groups express sulfakinin-immunoreactive material. Extensive arborization of dendrites containing sulfakinin-like peptides occurs within the neuropile of both species, suggesting a neurotransmitter/neuromodulator function. In the cockroach, there is clear evidence of direct distribution of sulfakinin-like peptides along axons to the foregut tissue, and a plexus of retrocerebral nerves is likely to serve as a neurohaemal release site. Neurohaemal release into the dorsal aorta is also postulated. Sulfakinin-immunoreactive axons do not innervate the hindgut in either cockroaches or crickets. Sulfakinin may function as a gut myotropin in the Blattodea, in addition to functioning as a neurotransmitter within the central nervous system. This latter function appears to be general across insect orders, while the neurohaemal distribution and myotropic activity are restricted to the Blattodea.

Evidence for gene conversion between tandemly duplicated cytoplasmic actin genes of Helicoverpa armigera (Lepidoptera:Noctuidae)

Tandemly duplicated actin genes have been isolated from a Helicoverpa armigera genomic library. Sequence comparisons with actin genes from other species suggest they encode cytoplasmic actins, being most closely related to the Bombyx mori A3 actin gene. The duplicated H. armigera actin genes, termed A3a and A3b, share 98.3% nucleotide sequence identity over their entire putative coding region. Analysis of the distribution of nucleotide differences shows the first 763 bp are identical between the two coding regions, with the 18 nucleotide changes occurring in the remaining 366 bp. This observation suggests a gene conversion event has taken place between the duplicated H. armigera A3a and A3b actin genes. Translation of the open-reading frames indicates the products of these genes are identical, apart from a single amino acid difference at codon 273. Polymerase chain reaction and northern blot analysis have shown both H. armigera A3a and A3b genes are expressed during pupal development and in the brain of newly enclosed adults. A region 5' of the H. armigera A3a actin gene start codon has been identified which contains regulatory sequences commonly found in the promoter region of actin genes, including TATA, CAAT, and CArG motifs.

Lepidopteran peptides of the allatostatin superfamily.

Peptides of the allatostatin superfamily with the C-terminal amino acid sequence -YXFGL-NH2 have been isolated and identified from the lepidopterans, the codling moth, Cydia pomonella (Tortricidae) and the bollworm, Helicoverpa armigera (Noctuidae). The peptides, designated cydiastatins and helicostatins respectively, were monitored during purification with radioimmunoassays based on the callatostatins of the blowfly Calliphora vomitoria. The eight peptides from each of the two species appear to form an homologous series with four identical and three that differ by a single amino acid. This study demonstrates the ubiquitous nature of this family of peptides in insects.

Identification of the dipteran Leu-callatostatin peptide family: the pattern of precursor processing revealed by isolation studies in Calliphora vomitoria.

Information from the Leu-callatostatin gene sequences of the blowflies Calliphora vomitoria and Lucilia cuprina was used to develop antisera specific for the variable post-tyrosyl amino-acid residues Ser, Ala and Asn of the common Leu-callatostatin C-terminal pentapeptide sequence -YXFGL-NH2. Radioimmunoassays based on these antisera were used to purify peptides from an extract of 40000 blowfly heads. Five neuropeptides of the Leu-callatostatin family were identified. Three have a seryl residue in the post-tyrosyl position. Two of these are octapeptides that differ only at the N-terminal residue; NRPYSFGL-NH2 and ARPYSFGL-NH2, whilst the third is the heptapeptide derived by N-terminal trimming; RPYSFGL-NH2. Two octapeptides in which X is Ala and Asn were also identified; VERYAFGL-NH2 and LPVYNFGL-NH2. The latter peptide is derived by processing at the internal dibasic site of a putative heneicosapeptide encoded by the DNA. These findings stress the necessity to have putative structures verified at the peptide level. Potent, reversible inhibitory effects on the spontaneous contractile activity of the blowfly rectum were recorded for ARPYSFGL-NH2 (monophasic dose-response curve with an IC50 = 10 fM) and for LPVYNFGL-NH2 (biphasic dose-response curve with IC50 values of approximately 1 fM and 1 nM). It is suggested that regulation of gut motility in insects, rather than an allatostatic function, may represent an ancestral and universal function of the allatostatins. One of the reasons for the large number of members of the Leu-callatostatin family appears to be in the provision of an integrated form of gut motility control, with different peptides controlling specific regions of the gut.

Isolation of alpha cluster esterase genes associated with organophosphate resistance in Lucilia cuprina.

PCR primers designed from the alpha-esterase gene cluster of Drosophila melanogaster have been used to isolate fragments from eight esterase genes in the Australian sheep blowfly, Lucilia cuprina. Phylogenetic analysis suggests that three are homologues of the alpha E7, alpha E8 and alpha E9 genes of the alpha-esterase cluster of D. melanogaster. A further three are also probably alpha-esterases, whereas the remaining two more closely resemble beta-esterases. Transcripts for five of the alpha-esterase genes were detected by PCR in adult midgut, consistent with a role for these enzymes in digestion and/or detoxification. Based on the tissue distribution of these transcripts, Lc alpha E7 may possibly encode the esterase, E3, which is involved in organophosphate resistance.

The fate of a thiazolidinedione antidiabetic agent in rat and dog.

1. The fate of [14C]BRL 49653C, a novel thiazolidinedione antidiabetic agent, has been studied following oral administration to the rat and dog. 2. Clearance was almost exclusively by metabolism, with only small amounts of unchanged BRL 49653 being excreted by either species. 3. Phase I metabolism resulted in ring hydroxylation, N-demethylation and oxidative removal of the pyridinylamino function to yield a phenoxyacetic acid derivative. 4. Sulphation of phase I metabolites occurred in both species, but glucuronidation was only observed in the rat. 5. The parent compound was the major circulating component in both species at early times, but at later times sulphate conjugates of phase 1 metabolites were predominant.

The sulfakinins of the blowfly Calliphora vomitoria. Peptide isolation, gene cloning and expression studies.

The nonapeptide, Phe-Asp-Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2 was isolated from heads of the blowfly Calliphora vomitoria. Designated callisulfakinin I, the peptide is identical to the earlier known drosulfakinin I of Drosophila melanogaster and to neosulfakinin I of Neobellieria bullata. It belongs to the sulfakinin family, all known members of which (from flies, cockroaches and locusts) have the C-terminal heptapeptide sequence Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2. The callisulfakinin gene of C. vomitoria was cloned and sequenced. In addition to callisulfakinin I, the DNA revealed a coding sequence for the putative tetradecapeptide. Gly-Gly-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His- Met-Arg-Phe-NH2, callisulfakinin II. However, this peptide was not identified in the fly head extracts. Confocal laser scanning immunocytochemical studies with antisera raised against the synthetic undecapeptide C-terminal fragment of drosulfakinin II from D. melanogaster, Asp-Gln-Phe-Asp-Asp-Tyr(SO3)- Gly-His-Met-Arg-Phe-NH2, revealed only four pairs of sulfakinin neurones in the brain of C. vomitoria and no others anywhere else in the neural, endocrine or gut tissues. In situ hybridisation studies with a digoxigenin-labelled sulfakinin gene probe (from the blowfly Lucilia cuprina) also revealed only four pairs of neurones in the brain. The perikarya of two pairs of cells are situated medially in the caudo-dorsal region, close to the roots of the ocellar nerve. The other perikarya are slightly more posterior and lateral. Although it has been suggested by several authors that the insect sulfakinins are homologous to the vertebrate peptides gastrin and cholecystokinin, such arguments (based essentially on C-terminal structural similarities) do not take account of important differences in the C-terminal tetrapeptide. His-Met-Arg-Phe-NH2 in the sulfakinins, compared with Trp-Met-Asp-Phe-NH2 in gastrin and cholecystokinin. Furthermore, whereas the sulfakinin neurons of C. vomitoria are small in number and have a very specialised location, a greater number of cells throughout the nervous system react positively to gastrin/cholecystokinin antisera. Chromatographic profiles of the present study also revealed peaks of gastrin/cholecystokinin-immunoreactive material separate from the sulfakinin peptides. This evidence suggests that the insect and vertebrate peptides may not necessarily be homologous.

Leu-callatostatin gene expression in the blowflies Calliphora vomitoria and Lucilia cuprina studied by in situ hybridisation: comparison with Leu-callatostatin confocal laser scanning immunocytochemistry.

In situ hybridisation studies using a digoxigenin-labelled DNA probe encoding the Leu-callatostatin prohormone of the blowflies Calliphora vomitoria and Lucilia cuprina have revealed a variety of neurones in the brain and thoracico-abdominal ganglion, peripheral neurosecretory neurones, and endocrine cells of the midgut. With two exceptions, the hybridising cells are the same as those previously identified in immunocytochemical studies of sections and whole-mounts using Leu-callatostatin COOH-terminal-specific antisera. Within the brain and suboesophageal ganglion, there is a variety of neurones ranging from a single pair of large cells situated in the dorsal protocerebrum, to the several pairs of neurones in the tritocerebrum, some of which, in immunocytochemical preparations, can be seen to project via axons in the cervical connective to the thoracico-abdominal ganglion. In the medulla of the optic lobes, numerous small interneurones hybridise with the probe, as do clusters of similar-sized neurones close to the roots of the ocellar nerves. These results indicate that the Leu-callatostatin neuropeptides of the brain play a variety of roles in neurotransmission and neuromodulation. There are only three pairs of Leu-callatostatin-immunoreactive neurones in the thoracico-abdominal ganglion, at least two pairs of which project axons along the median abdominal nerve to provide extensive innervation of the hindgut. The Leu-callatostatin peripheral neurosecretory cells are located in close association with both nerve and muscle fibres in the thorax. In addition to neuronal Leu-callatostatin, the presence of the peptide and its mRNA has been demonstrated in endocrine cells in the posterior part of the midgut. These observations provide an example of a named brain/gut peptide in an insect.

endAFS, a novel family E endoglucanase gene from Fibrobacter succinogenes AR1.

The complete nucleotide sequence of endAFS, an endoglucanase gene isolated from the ruminal anaerobe Fibrobacter succinogenes AR1, was determined. endAFS encodes two overlapping open reading frames (ORF1 and ORF2), and it was proposed that a -1 ribosomal frameshift was required to allow contiguous synthesis of a 453-amino-acid endoglucanase. A proline- and threonine-rich region at the C terminus of ORF1 and rare codons for arginine and threonine were coincident with the proposed frameshift site. ENDAFS is proposed to be a member of subgroup 1 of family E endoglucanases, of which endoglucanases from Thermomonospora fusca and Persea americana (avocado) are also members. Endoglucanases from Clostridium thermocellum and Pseudomonas fluorescens form subgroup 2.

Characterization of the AdhSL regulatory mutation in Drosophila melanogaster.

We describe the characterization of a previously reported control mutation, AdhSL, in the alcohol dehydrogenase gene of Drosophila melanogaster, which results in decreased production of ADH molecules and subsequently lower ADH activity in adults. We find that the regulatory element modifies ADH mRNA levels and acts cis on both ADH protein and mRNA. It is not promoter specific but is developmentally specific to the adult stage. The AdhSL allele carries a 4.5-kb insert approximately 3 kb 5' to the distal promoter. This new insertion may be responsible for the regulatory phenotype of AdhSL.

Frequency and temperature dependent selection at the alcohol dehydrogenase-1 locus of Drosophila buzzatii.

Genotype frequencies at the alcohol dehydrogenase-1 (Adh-1) locus of D. buzzatii were analysed for deviations from Hardy-Weinberg equilibria in the progeny of laboratory populations established at five initial Adh-1b allele frequencies and kept at either 18 degrees C, 25 degrees C or 30 degrees C. At 25 degrees C, no observed genotype frequencies were significantly different from Hardy-Weinberg expectations. Observed frequencies of heterozygotes were generally less than expected for populations at 18 degrees C and 30 degrees C. Fitness differences among genotypes were greater in males than in females, with Adh-1b homozygotes having highest fitness at 18 degrees C and Adh-1c homozygotes having highest fitness at 30 degrees C. The results are discussed in relation to previous field and laboratory studies on D. buzzatii and to the Adh polymorphism of D. melanogaster.

Temporal and microgeographic variation in allozyme frequencies in a natural population of Drosophila buzzatii.

Temporal variation in allozyme frequencies at six loci was studied by making monthly collections over 4 yr in one population of the cactophilic species Drosophila buzzatii. Ten sites were defined within the study locality, and for all temporal samples, separate collections were made at each of these sites. Population structure over microgeographic space and changes in population structure over time were analyzed using F-statistic estimators, and multivariate analyses of allele and genotype frequencies with environmental variables were carried out. Allele frequencies showed significant variation over time, although there were no clear cyclical or seasonal patterns. A biplot analysis of allele frequencies over seasons within years and over years showed clear discrimination among years by alleles at four loci. During the 4 yr, three alleles showed directional changes which were associated with directional changes in environmental variables. Significant associations with one or more environmental variables were found for allele frequencies at every locus and for both expected and observed heterozygosities (except those for Est-1 and Est-2). Thus, variation in allele frequencies over time cannot be attributed solely to drift. Significant linkage disequilibria were detected among three loci (Est-2, Hex and Aldox), but there was no evidence for spatial or temporal patterns. The F-statistic analyses showed significant differentiation among months within years for all loci, but the statistic used (coancestry) was heterogeneous among loci. Estimates of F (inbreeding) for all loci were significantly different from zero, with the loci in four groups, Adh-1 (negative), Pgm(small positive), Est-2 and Hex (intermediate) and Est-1 and Aldox (high positive). The correlation of genes within individuals within populations (f) for each locus in each month by site sample differed among loci, as did the (f) for each locus in each month by site sample differed among loci, as did the patterns of change in f over time (seasons). Heterogeneity in the F-statistic estimates indicates that natural selection is directly or indirectly affecting allele and genotype frequencies at some loci. However, the F-statistic analyses showed essentially no microgeographic structure (i.e., among sites), although there was significant heterogeneity in allele frequencies among flies emerging from individual rots. Thus, microspatial heterogeneity probably is most important at the level of individual rots, and coupled with habitat selection, it could be a major factor promoting diversifying selection and the maintenance of polymorphism.(ABSTRACT TRUNCATED AT 400 WORDS)

Allozyme genotypes of Drosophila buzzatii: feeding and oviposition preferences for microbial species, and habitat selection.

Mature, mated female D. buzzatii were given a choice of nine microbial communities actively growing on cactus homogenate in laboratory population cages, and tests were made to determine if flies of different genotypes (for seven allozyme loci) chose different microorganism species for either feeding or oviposition. Variation in feeding preferences was determined from assays of electrophoretic genotypes and the ingested microorganism species of individual flies. Oviposition preference variation was analyzed indirectly by assaying the genotypes of individuals raised from eggs laid on different microorganisms. No significant evidence was found for differences in feeding preferences among adults of different genotypes. For oviposition preferences, there were significant microorganism-genotype associations for each of seven polymorphic loci. Analyses of the total electrophoretic genotype, rather than of individual loci, showed that the genotypes of eggs laid on the same microorganism species were more similar than those laid on different species. That is, females of different genotypes show habitat selection for oviposition sites, which would facilitate the maintenance of genetic polymorphisms.

The ecology of the yeast flora in necroticOpuntia cacti and of associatedDrosophila in Australia.

A survey was made of the yeast communities isolated from necrotic tissue of 4 species of prickly-pear cacti (Opuntia stricta, O. tomentosa, O. monacantha, andO. streptacantha) which have colonized in Australia. Yeast communities were sampled from a number of localities and at different times. Cactus specific yeasts accounted for 80% of the total isolates, and the 3 most common species contributed 63% of the total. Comparisons of the species compositions of the yeast communities indicated that the differences among communities were greater betweenOpuntia species than between different localities within a single cactus species, and also that differences between years were greater than average differences between localities within years. Multivariate statistical tests of association between yeast community and physical features of rots indicated that temperature, pH, and age of rot all exerted some influence on the structure of the yeast community. Similar analyses involvingDrosophila species inhabiting these cactus rots suggested the existence of complex associations betweenDrosophila community, yeast community, and physical and chemical attributes of the cactus necroses.