Pheromones and Pheromone Receptors Are the Primary Determinants of Mating Specificity in the Yeast Saccharomyces Cerevisiae

Saccharomyces cerevisiae has two haploid cell types, a and alpha, each of which produces a unique set of proteins that participate in the mating process. We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity. We show that the capacity to synthesize alpha-factor pheromone and a-factor receptor is sufficient to allow mating by mat alpha 1 mutants, mutants that normally do not express any alpha- or a-specific products. Likewise, the capacity to synthesize a-factor receptor and alpha-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells. Thus, the a-factor receptor and alpha-factor pheromone constitute the minimum set of alpha-specific proteins that must be produced to allow mating as an alpha cell. Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat alpha 2 mutants, cells that simultaneously express both pheromones and both receptors. We created a series of strains that express different combinations of pheromones and receptors in a mat alpha 2 background. These constructions reveal that mat alpha 2 mutants can be made to mate as either a cells or as alpha cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type. Moreover, these studies show that the inability of mat alpha 2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to alpha factor by the a-factor receptor.     

Pheromone production in bark beetles

The first aggregation pheromone components from bark beetles were identified in 1966 as a mixture of ipsdienol, ipsenol and verbenol. Since then, a number of additional components have been identified as both aggregation and anti-aggregation pheromones, with many of them being monoterpenoids or derived from monoterpenoids. The structural similarity between the major pheromone components of bark beetles and the monoterpenes found in the host trees, along with the association of monoterpenoid production with plant tissue, led to the paradigm that most if not all bark beetle pheromone components were derived from host tree precursors, often with a simple hydroxylation producing the pheromone. In the 1990s there was a paradigm shift as evidence for de novo biosynthesis of pheromone components began to accumulate, and it is now recognized that most bark beetle monoterpenoid aggregation pheromone components are biosynthesized de novo. The bark beetle aggregation pheromones are released from the frass, which is consistent with the isoprenoid aggregation pheromones, including ipsdienol, ipsenol and frontalin, being produced in midgut tissue. It appears that exo-brevocomin is produced de novo in fat body tissue, and that verbenol, verbenone and verbenene are produced from dietary α-pinene in fat body tissue. Combined biochemical, molecular and functional genomics studies in Ips pini yielded the discovery and characterization of the enzymes that convert mevalonate pathway intermediates to pheromone components, including a novel bifunctional geranyl diphosphate synthase/myrcene synthase, a cytochrome P450 that hydroxylates myrcene to ipsdienol, and an oxidoreductase that interconverts ipsdienol and ipsdienone to achieve the appropriate stereochemistry of ipsdienol for pheromonal activity. Furthermore, the regulation of these genes and their corresponding enzymes proved complex and diverse in different species. Mevalonate pathway genes in pheromone producing male I. pini have much higher basal levels than in females, and feeding induces their expression. In I. duplicatus and I. pini, juvenile hormone III (JH III) induces pheromone production in the absence of feeding, whereas in I. paraconfusus and I. confusus, topically applied JH III does not induce pheromone production. In all four species, feeding induces pheromone production. While many of the details of pheromone production, including the site of synthesis, pathways and knowledge of the enzymes involved are known for Ips, less is known about pheromone production in Dendroctonus. Functional genomics studies are under way in D. ponderosae, which should rapidly increase our understanding of pheromone production in this genus. This chapter presents a historical development of what is known about pheromone production in bark beetles, emphasizes the genomic and post-genomic work in I. pini and points out areas where research is needed to obtain a more complete understanding of pheromone production.     

The enemy of my enemy is still my enemy: competitors add to predator load of a tree‐killing bark beetle

1 The mountain pine beetle Dendroctonus ponderosae is a major tree‐killing bark beetle in North America. We evaluated how the subsequent arrival of a competing bark beetle Ips pini influences the arrival of predators and their impact on both species. 2 The predators Temnochila chlorodia and Enoclerus sphegeus were strongly attracted to pheromones of D. ponderosae. By contrast, Enoclerus lecontei was mostly attracted to I. pini pheromones. The host compound myrcene synergized attraction of both D. ponderosae and E. sphegeus to the pheromone of D. ponderosae. However, it inhibited attraction of both I. pini and E. lecontei to I. pini’s pheromone. 3 Dendroctonus ponderosae were more attracted to trees than logs treated with its pheromones, whereas I. pini were more attracted to logs than trees treated with its pheromones. Some 78% of T. chlorodia were captured at hosts baited with D. ponderosae pheromones, whereas 83% of E. lecontei were captured at hosts baited with I. pini pheromones. We characterized the sequence of arrival to live trees baited with pheromones of D. ponderosae as: D. ponderosae, T. chlorodia, E. sphegeus, I. pini, E. lecontei. 4 Various combinations of I. pini and predators were added to logs colonized by D. ponderosae in the above sequence of arrival observed in live trees baited with D. ponderosae aggregation pheromones. Ips pini reduced D. ponderosae adult brood production. However, the combination of I. pini and E. lecontei did not raise D. ponderosae brood production above that observed with only I. pini present. Similarly, the combination of I. pini and T. chlorodia did not reduce D. ponderosae brood production below that observed with I. pini alone. By contrast, the combination of I. pini, T. chlorodia and E. lecontei caused more brood loss to D. ponderosae than I. pini alone. 5 Enoclerus lecontei did not reduce brood production by T. chlorodia, whereas T. chlorodia substantially reduced brood production by E. lecontei. 6 Secondary bark beetles that exploit the resource created by primary tree‐killing species exert negative effects through both competition and increased predator load. Implications to the population dynamics, ecology and evolution of tree‐killing bark beetles are discussed.     

The mode of evolution of aggregation pheromones in Drosophila species

Aggregation pheromones are used by fruit flies of the genus Drosophila to assemble on breeding substrates, where they feed, mate and oviposit communally. These pheromones consist of species-specific blends of chemicals. Here, using a phylogenetic framework, we examine how differences among species in these pheromone blends have evolved. Theoretical predictions, genetic evidence, and previous empirical analysis of bark beetle species, suggest that aggregation pheromones do not evolve gradually, but via major, saltational shifts in chemical composition. Using pheromone data for 28 species of Drosophila we show that, unlike with bark beetles, the distribution of chemical components among species is highly congruent with their phylogeny, with closely related species being more similar in their pheromone blends than are distantly related species. This pattern is also strong within the melanogaster species group, but less so within the virilis species group. Our analysis strongly suggests that the aggregation pheromones of Drosophila exhibit a gradual, not saltational, mode of evolution. We propose that these findings reflect the function of the pheromones in the ecology of Drosophila, which does not hinge on species specificity of aggregation pheromones as signals.     

Frontalin: De novo biosynthesis of an aggregation pheromone component by Dendroctonus spp. bark beetles (Coleoptera: Scolytidae)

The pheromone component, frontalin (1,5-dimethyl-6,8-dioxabicyclo[3.2.1]octane) is thought to be formed in Dendroctonus spp. bark beetles through the cyclization of oxygenated 6-methyl-6-hepten-2-one (6-MHO). Unlike many of the isoprenoid pheromone components of bark beetles, there is no obvious immediate host conifer precursor for 6-MHO or frontalin. To elucidate the biosynthetic pathway of frontalin, juvenile hormone-treated male Dendroctonus jeffreyi were injected separately with [1-(14)C]acetate, [2-(14)C]mevalonolactone, [1-(14)C]isopentenol, [1-(14)C]:[1-(3)H]isopentenol, and [4,5-(3)H]leucine. Subsequently volatiles were collected on Porapak Q from these males and abdominal tissues were extracted. Radio-HPLC analyses of extracts from males injected with each radiolabeled substrate showed that radioactivity from the injected precursors eluted in a peak with a retention time that matches that of unlabeled frontalin. In all cases, HPLC fractions containing radiolabel that eluted at the same time as a frontalin standard were analyzed by GC-FID and GC-MS to confirm the presence of frontalin. In a separate study, male D. jeffreyi were injected with [1-(13)C]acetate and an abdominal tissue extract from these insects was analyzed by tandem gas chromatography-isotope ratio monitoring-mass spectrometry (GC-IRM-MS), which unequivocally showed incorporation of (13)C into frontalin. Because mevalonate is the key intermediate in the isoprenoid pathway, its incorporation (as mevalonolactone) into frontalin provides compelling evidence that the biosynthesis of frontalin involves that pathway in some form. In the experiment with [1-(14)C]:[1-(3)H]isopentenol, there was no significant difference in the mean percentage incorporation of either radioisotope into frontalin. This supports the role of the classical isoprenoid pathway, as tritium would be lost if only a hybrid pathway were involved. Confirming that de novo synthesis may be general to all Dendroctonus spp., (14)C-acetate was also incorporated into frontalin by females of D. rufipennis and D. simplex. A radiolabeled precursor/pathway inhibitor study showed that the fatty acid synthase inhibitor, 2-octynoic acid, increased (although not significantly) the mass of frontalin produced and significantly increased the percentage incorporation of radioactivity from [1-(14)C]acetate into frontalin. This suggests that as fatty acid biosynthesis is blocked, an increased amount of acetate is funneled into frontalin production via the isoprenoid pathway.     

The ultrastructural investigation of the midgut in the quill mite Syringophilopsis fringilla (Acari,Trombidiformes: Syringophilidae)

The midgut of the females of Syringophilopsis fringilla (Fritsch) composed of anterior midgut and excretory organ (=posterior midgut) was investigated by means of light and transmission electron microscopy. The anterior midgut includes the ventriculus and two pairs of midgut caeca. These organs are lined by a similar epithelium except for the region adjacent to the coxal glands. Four cell subtypes were distinguished in the epithelium of the anterior midgut. All of them evidently represent physiological states of a single cell type. The digestive cells are most abundant. These cells are rich in rough endoplasmic reticulum and participate both in secretion and intracellular digestion. They form macropinocytotic vesicles in the apical region and a lot of secondary lysosomes in the central cytoplasm. After accumulating various residual bodies and spherites, the digestive cells transform into the excretory cells. The latter can be either extruded into the gut lumen or bud off their apical region and enter a new digestive cycle. The secretory cells were not found in all specimens examined. They are characterized by the presence of dense membrane-bounded granules, 2–4 μm in diameter, as well as by an extensive rough endoplasmic reticulum and Golgi bodies. The ventricular wall adjacent to the coxal glands demonstrates features of transporting epithelia. The cells are characterized by irregularly branched apical processes and a high concentration of mitochondria. The main function of the excretory organ (posterior midgut) is the elimination of nitrogenous waste. Formation of guanine-containing granules in the cytoplasm of the epithelial cells was shown to be associated with Golgi activity. The excretory granules are released into the gut lumen by means of eccrine or apocrine secretion. Evacuation of the fecal masses occurs periodically. Mitotic figures have been observed occasionally in the epithelial cells of the anterior midgut.     

Disruptive selection maintains variable pheromone blends in the bark beetle Ips pini

The presence of heritable variation is a prerequisite for evolution, but natural selection typically reduces genetic variation. Variation can be maintained in traits under selection through spatial or temporal variation in fitness surfaces, frequency-dependent selection, or disruptive selection. We evaluated the maintenance of variation in the enantiomeric blend of pheromones employed by the bark beetle Ips pini (Say). In natural populations, we quantified fitness surfaces for mating success and progeny production. We investigated the effects of paternal pheromone blend on offspring survival by comparing the spatial scales at which pheromone blends and larval mortality agents vary. Males with extreme pheromone blends obtained up to 1.8 times as many mates who each laid equivalent numbers of eggs, producing strong disruptive selection on male pheromone blend. In combination with imperfect assortative mating that continually produces intermediate genotypes, this fitness surface is sufficient to maintain variation in a heritable trait that is strongly linked to fitness. The ultimate explanation for female preference is unknown but could be because of selection for reduced mortality from specialist predators that prefer common prey pheromone blends. Selection is most likely occurring at the scale of small resource patches within pine stands. Selection at coarser scales (pine stands) is unlikely because pheromone blends did not vary among pine stands. Selection at finer scales (within logs) is unlikely because males of similar enantiomeric blends were not aggregated on logs, and male pheromone blend did not affect the spacing to neighboring galleries. This study documents a rare case of diversifying selection in natural populations.     

Parasitoids and dipteran predators exploit volatiles from microbial symbionts to locate bark beetles

Host location by parasitoids and dipteran predators of bark beetles is poorly understood. Unlike coleopteran predators that locate prey by orienting to prey pheromones, wasps and flies often attack life stages not present until after pheromone production ceases. Bark beetles have important microbial symbionts, which could provide sources of cues. We tested host trees, trees colonized by beetles and symbionts, and trees colonized by symbionts alone for attractiveness to hymenopteran parasitoids and dipteran predators. Field studies were conducted with Ips pini in Montana. Three pteromalid wasps were predominant. All were associated with the second and third instars of I. pini. Heydenia unica was more attracted to logs colonized by either I. pini or the fungus Ophiostoma ips than logs alone or blank controls (screen with no log). Rhopalicus pulchripennis was more attracted to logs colonized by I. pini than logs alone or blank controls. Dibrachys cavus was attracted to logs but did not distinguish whether or not they were colonized. Two dolichopodid predators were predominant. A Medetera species was more attracted to colonized than uncolonized logs and more attracted to logs than blank controls. It was also more attracted to logs colonized with the yeast Pichia scolyti than uncolonized logs, but attraction was less consistent. An unidentified dolichopodid was more attracted to logs colonized with I. pini, O. ips, and the bacteria Burkholderia sp., than to uncolonized logs. It was also attracted to uncolonized logs. Its responses were less consistent and pronounced than H. unica. These results suggest some parasitoids and dipteran predators exploit microbial symbionts of bark beetles to locate hosts. Overall, specialists showed strong attraction to fungal cues, whereas generalists were more attracted by plant volatiles. These results also show how microbial symbionts can have conflicting effects on host fitness.     

Multispecies trapping of six pests of scots pine in Sweden and Poland

Abstract:  Multispecies sex pheromone trapping (trapping of more than one species in the same trap) for the pine shoot moth Rhyacionia buoliana , the pine moth, Dendrolimus pini , the nun moth, Lymantria monacha , the pine beauty moth, Panolis flammea and the pine sawflies Diprion pini and Neodiprion sertifer was evaluated. The catch from traps baited with the pheromone of a single species was compared with the catch from traps baited with pheromones for several species. The catch in the multispecies traps was significantly reduced in comparison with the single species traps for Dendrolimus pini , L. monacha and N. sertifer . Neodiprion sertifer was most likely inhibited by the Diprion pini pheromone. A follow-up study of Dendrolimus pini and L. monacha showed no pheromonal interference between them. Further studies are needed to evaluate the feasibility of multispecies monitoring. However, for practical applications a slight decrease in catch, due to pheromonal interference, could probably be tolerated as long as the catch reflects total population density.     

Identification and Characterization of a Determinant (eep) on the Enterococcus faecalis Chromosome That Is Involved in Production of the Peptide Sex Pheromone cAD1

Plasmid-free strains of Enterococcus faecalis secrete a peptide sex pheromone, cAD1, which specifically induces a mating response by donors carrying the hemolysin plasmid pAD1 or related elements. A determinant on the E. faecalis OG1X chromosome has been found to encode a 46.5-kDa protein that plays an important role in the production of the extracellular cAD1. Wild-type E. faecalis OG1X cells harboring a plasmid chimera carrying the determinant exhibited an eightfold enhanced production of cAD1, and plasmid-free cells carrying a mutated chromosomal determinant secreted undetectable or very low amounts of the pheromone. The production of other pheromones such as cPD1, cOB1, and cCF10 was also influenced, although there was no effect on the pheromone cAM373. The determinant, designated eep (for enhanced expression of pheromone), did not include the sequence of the pheromone. Its deduced product (Eep) contains apparent membrane-spanning sequences; conceivably it is involved in processing a pheromone precursor structure or in some way regulates expression or secretion.     

Influence of sex,maturity and host substances on pheromones in the guts of the bark beetles, Ips paraconfusus and Dendroctonus brevicomis

Pheromones and metabolites of host (ponderosa pine) compounds were found in association with the hindgut of both naturally fed and of non-fed, host vapour-exposed bark beetles, Ips paraconfusus and Dendroctonus brevicomis. Much smaller amounts were found in the corresponding heads and mid guts. Sex-specific differences in content of pheromones were observed as in earlier studies. Exposure of I. paraconfusus to vapours of a pheromone component, ipsenol and other monoterpene alcohols resulted in their accumulation in the hindgut but relatively very low amounts in the head. The possible sites of pheromone biosynthesis are discussed. Exposure of male I. paraconfusus to vapours of host compounds, myrcene and α-pinene, revealed that immature adults do not produce the pheromone components, ipsenol and ipsdienol, as mature adults do while both immature and mature sexes produced another pheromone component, cis-verbenol, as well as trans-verbenol and myrtenol. Immature D. brevicomis adults did not contain pheromones until their exposure to vapours of (?)-α-pinene which caused production of trans-verbenol but only about 10% that of mature adults treated similarly. Verbenone, a male-produced inhibitory pheromone of D. brevicomis, apparently was not synthesized from (?)-α-pinene in females nor was its synthesis in males enhanced by exposure to this host compound.     

Molecular mechanisms underlying sex pheromone production in the silkmoth, Bombyx mori: characterization of the molecular components involved in bombykol biosynthesis

Many species of female moths produce sex pheromones to attract conspecific males. To date, sex pheromones from more than 570 moth species have been chemically identified. Most moth species utilize Type I pheromones that consist of straight-chain compounds 10-18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester and usually with several double bonds. In contrast, some moth species use unsaturated hydrocarbons or hydrocarbon epoxides, classified as Type II lepidopteran pheromones, as sex pheromones. Studies over the past three decades have demonstrated that female moths usually produce sex pheromones as multi-component blends where the ratio of the individual components is precisely controlled, thus making it possible to generate species-specific pheromone blends. As for the biosynthesis of Type I pheromones, it is well established that they are de novo synthesized in the pheromone gland (PG) through modifications of fatty acid biosynthetic pathways. However, as many of the molecular components within the PG cells (i.e., enzymes, proteins, and small regulatory molecules) have not been functionally characterized, the molecular mechanisms underlying sex pheromone production in PG cells remain poorly understood. To address this, we have recently characterized some of the molecules involved in the biosynthesis of the sex pheromone bombykol in the silkmoth, Bombyx mori. Characterization of these, and other, key molecules will facilitate our understanding of the precise mechanisms underlying lepidopteran sex pheromone production.     

Digestive enzyme compartmentalization and recycling and sites of absorption and secretion along the midgut of Dermestes maculatus (Coleoptera) larvae

Bostrichiformia is the less known major series of Coleoptera regarding digestive physiology. The midgut of Dermestes maculatus has a cylindrical ventriculus with anterior caeca. There is no cell differentiation along the ventriculus, except for the predominance of cells undergoing apocrine secretion in the anterior region. Apocrine secretion affects a larger extension and a greater number of cells in caeca than in ventriculus. Ventricular cells putatively secrete digestive enzymes, whereas caecal cells are supposed to secrete peritrophic gel (PG) glycoproteins. Feeding larvae with dyes showed that caeca are water-absorbing, whereas the posterior ventriculus is water-secreting. Midgut dissection revealed a PG and a peritrophic membrane (PM) covering the contents in anterior and posterior ventriculus, respectively. This was confirmed by in situ chitin detection with FITC-WGA conjugates. Ion-exchange chromatography of midgut homogenates, associated with enzymatic assays with natural and synthetic substrates and specific inhibitors, showed that trypsin and chymotrypsin are the major proteinases, cysteine proteinase is absent, and aspartic proteinase probably is negligible. Amylase and trypsin occur in contents and decrease along the ventriculus; the contrary is true for cell-membrane-bound aminopeptidase. Maltase is cell-membrane-bound and predominates in anterior and middle midgut. Digestive enzyme activities in hindgut are negligible. This, together with dye data, indicates that enzymes are recovered from inside PM by a posterior-anterior flux of fluid outside PM before being excreted. The combined results suggest that protein digestion starts in anterior midgut and ends in the surface of posterior midgut cells. All glycogen digestion takes place in anterior midgut.     

Queen-signal modulation of worker pheromonal composition in honeybees

Worker sterility in honeybees is neither absolute nor irreversible. Whether under queen or worker control, it is likely to be mediated by pheromones. Queen-specific pheromones are not exclusive to queens; workers with activated ovaries also produce them. The association between ovarian activation and queen-like pheromone occurrence suggests the latter as providing a reliable signal of reproductive ability. In this study we investigated the effect of queen pheromones on ovary development and occurrence of queen-like esters in workers' Dufour's gland. Workers separated from the queenright compartment by a double mesh behaved like queenless workers, activating their ovaries and expressing a queen-like Dufour's gland secretion, confirming that the pheromones regulating both systems are non-volatile. Workers with developed ovaries produced significantly more secretion than sterile workers, which we attribute primarily to increased ester production. Workers separated from the queenright compartment by a single mesh displayed a delayed ovarian development, which we attribute to interrupted transfer of the non-volatile pheromone between compartments. We suggest that worker expression of queen-like characters reflects a queen-worker arms race; and that Dufour's gland secretion may provide a reliable signal for ovarian activation. The associative nature between ovary development and Dufour's gland ester production remains elusive.     

Evidence for Gene Duplication and Allelic Codominance (not Hierarchical Dominance) at the Mating‐Type Locus of the Ciliate,Euplotes crassus

The high‐multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating‐type substance (pheromone). In strain L‐2D, now known to be homozygous at the mating‐type locus, we previously identified two pheromones (Ec‐α and Ec‐1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR‐73 characterized by a heterozygous genotype and strong mating compatibility with L‐2D strain. It was found that POR‐73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec‐α pheromone of L‐2D cells. The other two pheromones were shown to be new and were designated Ec‐2 and Ec‐3 to denote their structural homology with the Ec‐1 pheromone of L‐2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating‐type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes.     

Genetic and endocrine studies of the pregnancy-blocking pheromone of mice.

Secretion of the pregnancy-blocking pheromone was stimulated by injection of depo-testosterone cypionate into females and males of inbred strains of mice which do not normally secrete the pheromone. Testosterone treatment of SJL males altered pheromone secretion so that pregnancies were blocked when the stud male was of the same inbred strain; an event that does not normally occur. Injection of epiandrosterone, androstenedione, androsterone or testosterone significantly increased pheromone secretion in SJL females, but progesterone and dehydroepiandrosterone were ineffective. Kidney weights were significantly increased by administration of androgen metabolites and the possibility of the kidney being the site of pheromone synthesis is discussed. The preputial gland can be excluded as the site of pheromone synthesis since males which are hemizygous for the Tabby-J gene and have no preputial glands blocked pregnancies as effectively as their normal littermates. Preliminary results are also presented concerning the isolation of the pregnancy-blocking pheromone from urine. Urine was analysed by gas chromatography and a peak was observed whose concentration could be correlated with secretion of the pheromone, although the compound(s) has not been identified or tested for biological activity.     

Morphology and ultrastructure of the midgut in Piscicola geometra (Annelida, Hirudinea)

This paper presents information on the organization of the midgut and its epithelium ultrastructure in juvenile and adult specimens of Piscicola geometra (Annelida, Hirudinea), a species which is a widespread ectoparasite found on the body and gills and in the mouth of many types of fish. The analysis of juvenile nonfeeding specimens helped in the explanation of all alterations in the midgut epithelium which are connected with digestion. The endodermal portion (midgut) of the digestive system is composed of four regions: the esophagus, the crop, the posterior crop caecum, and the intestine. Their epithelia are formed by flat, cuboidal, or columnar digestive cells; however, single small cells which do not contact the midgut lumen were also observed. The ultrastructure of all of the regions of the midgut are described and discussed with a special emphasis on their functions in the digestion of blood. In P. geometra, the part of the midgut that is devoid of microvilli is responsible for the accumulation of blood, while the epithelium of the remaining part of the midgut, which has a distinct regionalization in the distribution of organelles, plays a role in its absorption and secretion. Glycogen granules in the intestinal epithelium indicate its role in the accumulation of sugar. The comparison of the ultrastructure of midgut epithelium in juvenile and adult specimens suggests that electron-dense granules observed in the apical cytoplasm of digestive cells take part in enzyme accumulation. Numerous microorganisms were observed in the mycetome, which is composed of two large oval diverticles that connect with the esophagus via thin ducts. Similar microorganisms also occurred in the cytoplasm of the epithelium in the esophagus, the crop, the intestine, and in their lumen. Microorganisms were observed both in fed adult and unfed juvenile specimens of P. geometra, which strongly suggests that vertical transmission occurs from parent to offspring.     

Fitness consequences of pheromone production and host selection strategies in a tree-killing bark beetle (Coleoptera: Curculionidae: Scolytinae)

Timing of arrival at a resource often determines an individual’s reproductive success. Tree-killing bark beetles can reproduce in healthy trees by attacking in adequate numbers to overcome host defences that could otherwise be lethal. This process is mediated by aggregation and antiaggregation pheromones. Beetles that arrive early in such a “mass attack” must contend with undiminished tree defences, and produce enough pheromones to attract more beetles, but have a head start on gallery construction and egg-laying. Beetles that arrive late may be impeded by competition and diminishing availability of phloem, but should experience fewer costs associated with pheromone production and battling tree defences. We investigated relationships between timing of arrival, body size, pheromone production and fitness in the southern pine beetle, Dendroctonus frontalis. In field experiments, we captured beetles that arrived early (pioneers) and late on slash pine trees, Pinus elliottii, and measured pheromone amounts in their hindguts. We marked gallery entrances of beetles as they landed on a tree and measured their reproductive success after the attack terminated. We found no difference in body size or pheromone amounts between early and late arrivers. Most beetles arrived at the middle of the attack sequence, and excavated longer galleries per day than early arrivers. The number of offspring produced per day by beetles that established galleries midway through mass attack was higher than those that arrived early or very late in the sequence. Our results suggest that beetles do not exhibit adaptive phenotypic plasticity in pre-landing pheromone production, depending on the extent of previous colonisation of a host. Rather, it appears that stabilising selection favours beetles that attack in the middle of the sequence, and contributes to attack synchrony. Synchronous attack on trees is essential before population booms characteristic of tree-killing bark beetles can occur in nature.     

Does the scent of a potential mate prevent the resorption of oocytes by apoptosis in Nauphoeta cinerea?

Abstract  We expect organisms to have evolved mechanisms to gather and use the information available within their environments, to steer resource investment decisions toward the traits that will give the greatest fitness returns. Pheromones are social signals, a common purpose of which is to act as indicators of mate presence. Consequently, pheromones have the potential to act as signals to increase or maintain reproductive investment over that of competing life-history traits. In the cockroach, Nauphoeta cinerea (Oliver), females pay costs of maintaining investment in reproduction when there are no males, and males produce pheromones that are known to effect female reproductive outcomes. Whether the pheromones have an influence on resource physiology is unknown. We subjected newly eclosed females to either a synthetic blend of male pheromones or control. We dissected females at 7, 12, 17 or 22 days. We measured apoptosis levels and size of all oocytes within the vitellerium, and measured dry fat body mass. Synthetic male pheromone blend did not have an effect on any measure of female reproductive or somatic resource physiology. Although negative results are always problematic, the success of the synthetic pheromone method in the past suggests that females may be insensitive to male pheromones in the pre-oviposition period, and may rely on mating stimulus rather than pheromone as the cue for oocyte maintenance and growth. Previous studies reporting effects of male pheromones on female reproduction suggest that the period of pheromone sensitivity may be between mating and birth.     

Functional expression of a bark beetle cytochrome P450 that hydroxylates myrcene to ipsdienol

The final steps in the pheromone-biosynthetic pathway of the pine engraver beetle, Ips pini (Say) (Coleoptera: Scolytidae) are unknown, but likely involve myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation to produce the aggregation pheromone component, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol). We have isolated a full-length I. pini cDNA encoding a cytochrome P450, CYP9T2. The recovered cDNA is 1.83kb and the open reading frame encodes a 532 amino acid protein. CYP9T2 is regulated by the same physiological factors that induce pheromone production. Quantitative real-time PCR experiments showed that feeding on host phloem induced CYP9T2 expression in males, but not females, and that basal expression levels are highest in male midguts, similar to other I. pini pheromone-biosynthetic genes. Microsomes prepared from Sf9 cells co-expressing baculoviral-mediated recombinant CYP9T2 and housefly (Musca domestica) NADPH-cytochrome P450 reductase converted myrcene to ipsdienol. The product identified by coupled GC-MS was mostly (4R)-(-)-ipsdienol, an important aggregation pheromone component for western North American I. pini. These results are consistent with CYP9T2 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway.