Selective maintenance of allozyme differences among sympatric host races of the apple maggot fly

Whether phytophagous insects can speciate in sympatry when they shift and adapt to new host plants is a controversial question. One essential requirement for sympatric speciation is that disruptive selection outweighs gene flow between insect populations using different host plants. Empirical support for host-related selection (i.e., fitness trade-offs) is scant, however. Here, we test for host-dependent selection acting on apple (Malus pumila)- and hawthorn (Crataegus spp.)-infesting races of Rhagoletis pomonella (Diptera: Tephritidae). In particular, we examine whether the earlier fruiting phenology of apple trees favors pupae in deeper states of diapause (or with slower metabolisms/development rates) in the apple fly race. By experimentally lengthening the time period preceding winter, we exposed hawthorn race pupae to environmental conditions typically faced by apple flies. This exposure induced a significant genetic response at six allozyme loci in surviving hawthorn fly adults toward allele frequencies found in the apple race. The sensitivity of hawthorn fly pupae to extended periods of warm weather therefore selects against hawthorn flies that infest apples and helps to maintain the genetic integrity of the apple race by counteracting gene flow from sympatric hawthorn populations. Our findings confirm that postzygotic reproductive isolation can evolve as a pleiotropic consequence of host-associated adaptation, a central tenet of nonallopatric speciation. They also suggest that one reason for the paucity of reported fitness trade-offs is a failure to consider adequately costs associated with coordinating an insect’s life cycle with the phenology of its host plant.

Tsetse thrombin inhibitor: Bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans

The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of Glossina morsitans morsitans (Diptera: Glossinidae). A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes. The full-length cDNA contains a 26-bp untranslated segment at its 5′ end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide. A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein. Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors. Recombinant tsetse thrombin inhibitor expressed in Escherichia coli and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity. The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal.

Jasmonate is essential for insect defense in Arabidopsis

The signaling pathways that allow plants to mount defenses against chewing insects are known to be complex. To investigate the role of jasmonate in wound signaling in Arabidopsis and to test whether parallel or redundant pathways exist for insect defense, we have studied a mutant (fad3–2 fad7–2 fad8) that is deficient in the jasmonate precursor linolenic acid. Mutant plants contained negligible levels of jasmonate and showed extremely high mortality (≈80%) from attack by larvae of a common saprophagous fungal gnat, Bradysia impatiens (Diptera: Sciaridae), even though neighboring wild-type plants were largely unaffected. Application of exogenous methyl jasmonate substantially protected the mutant plants and reduced mortality to ≈12%. These experiments precisely define the role of jasmonate as being essential for the induction of biologically effective defense in this plant–insect interaction. The transcripts of three wound-responsive genes were shown not to be induced by wounding of mutant plants but the same transcripts could be induced by application of methyl jasmonate. By contrast, measurements of transcript levels for a gene encoding glutathione S-transferase demonstrated that wound induction of this gene is independent of jasmonate synthesis. These results indicate that the mutant will be a good genetic model for testing the practical effectiveness of candidate defense genes.

Assembly of Lampbrush Chromosomes from Sperm Chromatin

We have examined the behavior of demembranated sperm heads when injected into the germinal vesicle (GV) of amphibian oocytes. Xenopus sperm heads injected into Xenopus GVs swelled immediately and within hours began to stain with an antibody against RNA polymerase II (Pol II). Over time each sperm head became a loose mass of chromosome-like threads, which by 24–48 h resolved into individually recognizable lampbrush chromosomes (LBCs). Although LBCs derived from sperm are unreplicated single chromatids, their morphology and immunofluorescent staining properties were strikingly similar to those of the endogenous lampbrush bivalents. They displayed typical transcriptionally active loops extending from an axis of condensed chromomeres, as well as locus-specific “landmarks.” Experiments with [3H]GTP and actinomycin D demonstrated that transcription was not necessary for the initial swelling of the sperm heads and acquisition of Pol II but was required for maintenance of the lampbrush loops. Splicing was not required at any stage during formation of sperm LBCs. When Xenopus sperm heads were injected into GVs of the newt Notophthalmus, the resulting sperm LBCs displayed very long loops with pronounced Pol II axes, like those of the endogenous newt LBCs; as expected, they stained with antibodies against newt-specific proteins. Other heterologous injections, including sperm heads of the frog Rana pipiens and the zebrafish Danio rerio in Xenopus GVs, confirm that LBCs can be derived from taxonomically distant organisms. The GV system should help identify both cis- and trans-acting factors needed to convert condensed chromatin into transcriptionally active LBCs. It may also be useful in producing cytologically analyzable chromosomes from organisms whose oocytes do not go through a typical lampbrush phase or cannot be manipulated by current techniques.

Discovery of genes involved with learning and memory: An experimental synthesis of Hirschian and Benzerian perspectives

The biological bases of learning and memory are being revealed today with a wide array of molecular approaches, most of which entail the analysis of dysfunction produced by gene disruptions. This perspective derives both from early “genetic dissections” of learning in mutant Drosophila by Seymour Benzer and colleagues and from earlier behavior-genetic analyses of learning and in Diptera by Jerry Hirsch and coworkers. Three quantitative-genetic insights derived from these latter studies serve as guiding principles for the former. First, interacting polygenes underlie complex traits. Consequently, learning/memory defects associated with single-gene mutants can be quantified accurately only in equilibrated, heterogeneous genetic backgrounds. Second, complex behavioral responses will be composed of genetically distinct functional components. Thus, genetic dissection of complex traits into specific biobehavioral properties is likely. Finally, disruptions of genes involved with learning/memory are likely to have pleiotropic effects. As a result, task-relevant sensorimotor responses required for normal learning must be assessed carefully to interpret performance in learning/memory experiments. In addition, more specific conclusions will be obtained from reverse-genetic experiments, in which gene disruptions are restricted in time and/or space.

A novel application of gene arrays: Escherichia coli array provides insight into the biology of the obligate endosymbiont of tsetse flies

Symbiotic associations with microorganisms are pivotal in many insects. Yet, the functional roles of obligate symbionts have been difficult to study because it has not been possible to cultivate these organisms in vitro. The medically important tsetse fly (Diptera: Glossinidae) relies on its obligate endosymbiont, Wigglesworthia glossinidia, a member of the Enterobacteriaceae, closely related to Escherichia coli, for fertility and possibly nutrition. We show here that the intracellular Wigglesworthia has a reduced genome size smaller than 770 kb. In an attempt to understand the composition of its genome, we used the gene arrays developed for E. coli. We were able to identify 650 orthologous genes in Wigglesworthia corresponding to ≈85% of its genome. The arrays were also applied for expression analysis using Wigglesworthia cDNA and 61 gene products were detected, presumably coding for some of its most abundant products. Overall, genes involved in cell processes, DNA replication, transcription, and translation were found largely retained in the small genome of Wigglesworthia. In addition, genes coding for transport proteins, chaperones, biosynthesis of cofactors, and some amino acids were found to comprise a significant portion, suggesting an important role for these proteins in its symbiotic life. Based on its expression profile, we predict that Wigglesworthia may be a facultative anaerobic organism that utilizes ammonia as its major source of nitrogen. We present an application of E. coli gene arrays to obtain broad genome information for a closely related organism in the absence of complete genome sequence data.

Isolation and identification of a diuretic hormone from the mealworm Tenebrio molitor.

A diuretic hormone of unusual structure was isolated from extracts of whole heads of the mealworm Tenebrio molitor. The hormone is a 37-aa peptide of 4371 Da, with the sequence SPTISITAPIDVLRKTWEQERARKQMVKNREFLNSLN. This peptide increases cAMP production in Malpighian tubules of T. molitor. The amino acid sequence reveals that this peptide is a member of the family of sauvagine/corticotropin-releasing factor/urotensin I-related insect diuretic hormones. The C-terminal sequence of this peptide is quite different from other members of this family, which have a hydrophobic C terminus (isoleucinamide or valinamide). When aligned comparably, T. molitor diuretic hormone has a more hydrophilic C terminus, leucylasparagine (free acid). In contrast to all other known diuretic hormones of this family, this peptide has exceptionally low stimulatory activity on cAMP production in Malpighian tubules of Manduca sexta. However, at nanomolar concentrations it stimulates cAMP production in Malpighian tubules of T. molitor. Diuretic hormones of this family have been isolated previously from Lepidoptera, Orthoptera, Dictyoptera, and Diptera. This appears to be the first diuretic hormone isolated from a coleopteran insect.