Differential toxicity of a phenolic glycoside from quaking aspen to Papilio glaucus butterfly subspecies,hybrids and backcrosses

Summary Papilio glaucus subspecies, hybrids and backcrosses exhibit greatly different abilities to use quaking aspen (Populus tremuloides) and other members of the Salicaceae as host plants. This study was conducted to test the hypotheses that phenolic glycosides account for the differences in larval performance, and that differential performance is correlated with differential larval esterase activities. To test the hypotheses we conducted first instar survival trials and fourth (penultimate) instar feeding trials with tremulacin, a phenolic glycoside. We also conducted assays of -glucosidase, esterase, and glutathione transferase activities, using midgut enzyme preparations from fifth instars. First instar survival on the tremulacin treated diet generally improved with a higher proportion of Papilio glaucus canadensis genes in the genotype, although survival in one backcross treatment was surprisingly low. Penultimate instars of P.g. glaucus and P.g. australis fed tremulacin treated black cherry leaves experienced a severe reduction in growth rate relative to larvae fed control leaves. This seriously suppressed growth was partially due to reduced consumption rates and reduced conversion efficiencies, however, approximate digestibility was not affected. In contrast, P. g. canadensis and hybrids showed no differences in growth rates between tremulacin treated and control leaves. Reciprocal backcrosses of hybrids with P. g. glaucus resulted in slightly suppressed growth on treated versus control leaves. The results suggest that after a certain threshold, increased proportions of P. g. glaucus genes resulted in poorer growth performance with tremulacin in the diet. Soluble esterase activities generally increased with the proportion of Papilio glaucus canadensis genes in the genotype, and paralleled overall trends in larval survival and feeding performance. We conclude that phenolic glycosides such as tremulacin are responsible for differential performance of Papilio glaucus subspecies, hybrids and backcrosses fed plants in the Salicaceae, and that detoxification of phenolic glycosides by midgut esterase explains why some Papilio glaucus genotypes can effectively utilize these plants.     

Differential responses of tiger swallowtail subspecies to secondary metabolites from tulip tree and quaking aspen

Summary Two subspecies of the eastern tiger swallowtail butterfly, Papilio glaucus, exhibit reciprocal inabilities to survive and grow on each other's preferred foodplant. P. g. canadensis R. & J. performs well on quaking aspen (Populus tremuloides Michx.) but not on tulip tree (Liriodendron tulipifera L.); P. g. glaucus L. performs well on tulip tree but not on quaking aspen. This study was designed to test the hypothesis that secondary metabolites in tulip tree and quaking aspen are responsible for these differential utilization abilities. We extracted and fractionated leaf constituents into different chemical classes, applied them to a mutually acceptable diet (black cherry, Prunus serotina, leaves), and bioassayed them against neonate larvae (survival) and penultimate instar larvae (survival, growth, digestibility and conversion efficiencies). For each plant species, one fraction in particular showed activity against the unadapted subspecies. One tulip tree fraction dramatically reduced survival of P. g. canadensis neonates, and reduced consumption rates, growth rates, and ECI's of fourth instar larvae. The tulip tree constituents most likely responsible for these effects are sesquiterpene lactones. One quaking aspen fraction greatly lowered survival of P. g. glaucus neonates, and decreased survival, consumption rates, growth rates and ECD's of fourth instar larvae. The compounds responsible for these results are probably simple phenols or phenolic glycosides. Surprisingly, P. g. glaucus and P. g. canadensis showed slightly poorer performance on the active tulip tree and quaking aspen fractions, respectively, indicating that even adapted insects incur a metabolic cost in the processing of their host's phytochemicals.     

POPULATION STRUCTURE AND HOST USE IN HYBRIDIZING SUBSPECIES OF PAPILIO GLAUCUS (LEPIDOPTERA: PAPILIONIDAE)

Two parapatric subspecies of the eastern tiger swallowtail butterfly, Papilio glaucus glaucus and P. g. canadensis, differ greatly in larval survival and growth on host plants in the Magnoliaceae, Salicaceae, and Betulaceae. In the first part of this study, butterflies were collected from 17 sites along a transect from Georgia to Quebec and used for allozyme electrophoresis. Two X- (or Z-)linked enzyme loci (Ldh and Pgd) showed allele frequency differences that delineated a hybrid zone between the subspecies in northern Pennsylvania and south-central New York. No significant linkage disequilibrium could be detected among allozyme loci within the hybrid zone samples. Genetic differentiation at Ldh and Pgd coincides with subspecies differences in diapause control and female mimicry, which are also sex-linked. Larval offspring of butterflies from 13 of the sites were then tested in the laboratory for survival and growth on Liriodendron tulipifera (Magnoliaceae), Populus deltoïdes (Salicaceae) and Betula lutea (Betulaceae). Steep clines in survival rates matched the position of the hybrid zone. Hybrid zone larvae showed intermediate survival rates and significant heterogeneity among families on all three plants, indicating presence of substantial genetic variation. The results suggest that differential host use by P. g. glaucus and P. g. canadensis is maintained primarily by independent clines in selection intensity for ability to use allopatric sets of host plants, coupled with restricted gene flow across the hybrid zone.     

Reciprocal latitudinal clines in oviposition behavior ofPapilio glaucus andP. canadensis across the Great Lakes hybrid zone: possible sex-linkage of oviposition preferences

Summary The eastern tiger swallowtail butterfly,Papilio glaucus, is the most polyphagous of all Papilionidae species. While diverse larval detoxication abilities are known for bothPapilio glaucus and the closely relatedP. canadensis, the factors affecting oviposition preferences in adult females are unknown. These congeneric species were studied to determine the extent of oviposition mistakes on toxic plants. We were also interested in comparing the geographic patterns of variation in oviposition preferences and the genetic basis of these differences. We conducted oviposition three-choice studies with the Canadian tiger swallowtail butterfly,Papilio canadensis, and the Eastern tiger swallowtail,Papilio glaucus, giving them the choice of (1) tuliptree,Liriodendron tulipifera, which is toxic to virtually all populations (P. canadensis) north of the Great Lakes Region of North America, (2) quaking aspen,Populus tremuloides, which is toxic to essentially all populations (P. glaucus) south of the Great Lakes, and (3) black cherry,Prunus serotina, which is an excellent foodplant for all members of thePapilio glaucus group, but which does not occur at latitudinal extremes of North America (in Alaska and most of Canada or the southern half of Florida). Handpaired interspecific hybrids were tested under the same experimental design to evaluate the possibility of sexlinked oviposition behavior. There was considerable variability in the choice of plants by individual butterflies, but a general trend suggesting that the females of each species had a lower preference for the plant toxic to their larvae. More than 6000 oviposition bouts were counted from 37 differentp. canadensis and 54p. glaucus females along a latitudinal transect of approximately 5000 km from Alaska south through the Great Lakes hybrid zone region to southern Florida. While not exceptionally high anywhere, the preference for aspen (Salicaceae) declined precipitously in central Michigan (45° N latitude) and remained very low (5–12%) in all locations southward to Florida, whereas we observed a reciprocal trend in preference for tuliptree (Magnoliaceae) which was greatest in Florida (87% of all eggs) and steadily declined northward across the Great Lakes region. Cherry was selected in these 3-choice tests at a relatively consistent and low frequency at all latitudes. Fixed allele differences in sex-linked (LDH and PGD) and autosomal (HK) electromorphs are known forP. glaucus andP. canadensis. Our electrophoretic data suggest that the preference of an individual female for aspen is not simply a characteristic of the northern species (P. canadensis) but can occur inP. glaucus females. The reciprocal situation is also evident in Northern Michigan and Wisconsin females (scored electrophoretically and morphologically asP. canadensis) which sometimes exhibit a clear preference for the toxic tuliptree. In fact, Alaskan populations ofP. canadensis chose tuliptree for about 52% of their eggs, even though none of their offspring has ever survived on this plant species in laboratory studies. We conclude that even with distinctive latitudinal trends, a considerable amount of local variation in relative oviposition preference exists among individuals of these polyphagous species. BothP. glaucus andP. canadensis will lay eggs on toxic plants. It appears that factors selecting against oviposition on toxic tuliptrees have been minimal (relative to other factors) in Alaska and somewhat stronger in the Great Lakes hybrid zone. It is in this zone of contact with tuliptree where selection against theP. canadensis populations ovipositing on tuliptree may be strong due to high larval mortality when such natural mistakes are made. We do not know whether behavioral preference changes evolutionarily preceded or followed the development of specific physiological detoxication abilities for tuliptree or quaking aspen. However, for bothP. canadensis andP. glaucus the occurrence of oviposition mistakes on toxic plants by adults extends geographically well beyond the larval detoxication abilities of their offspring. Hybrid female offspring of pairings with Michigancanadensis females andglaucus males show distinct preferences for tuliptree, suggesting that oviposition may be controlled by a factor (or factors) on the sex chromosome. Unfortunately we were unable to obtain reciprocal hybrids to evaluate the possibility of sex-linked aspen preference.     

Origins of the regional feeding abilities in the tiger swallowtail butterfly: ecological monophagy and the Papilio glaucus australis subspecies in Florida

Summary The Highlands County, Florida populations of the eastern tiger swallowtail butterfly, Papilio glaucus L., represent a putative subspecies, P. g. australis Maynard. The only foodplant ever reported in southern half of Florida is sweetbay, Magnolia virginiana L. (Magnoliaceae). In fact, there are apparently no other reported foodplants for the polyphagous tiger swallowtail species which exist in southern Florida. These ecologically monophagous P. g. australis populations were studied in order to determine whether or not significant behavioral and biochemical adaptations in the larvae are detectable as a result of the ecological specialization on sweetbay. These results suggest an emphatic yes. On sweetbay, neonate larval survival of these Florida populations is the best of any other population tested from across North America. In addition, penultimate instar larval growth rates are significantly faster on sweet bay than any other foodplant tested. Furthermore, these biochemical adaptations to sweetbay by P. g. australis in Florida are accompanied by a significantly poorer neonate survival and growth performance on northern foodplants (most notably the Salicaceae and Betulaceae). Reciprocal inabilities of the northern subspecies, P. g. canadensis, and the western P. rutulus to utilize sweetbay are described, as well as the reciprocal inabilities of two congeneric and sympatric (Florida) species, Papilio palamedes and Papilio troilus, that have specialized on another bay (redbay, Persea borbonia; of the Lauraceae). It remains uncertain to what degree such apparent negative genetic correlations in foodplant adaptations represent a driving force in the formation of host races or species, especially since so little is known about oviposition preferences of Papilio glaucus across North America. In Florida we have an apparently strict ecological monophagy coupled with a degree of reproductive isolation that has resulted in its tentative classification as a subspecies, P. g. australis. The role of foodplants in the speciation process can be significant even for P. glaucus, the most polyphagous of all 563 species of world Papilionidae. It remains uncertain whether the significant differences in detoxication and biochemical processing abilities observed in this study were evolved in parapatry or allopatry. Disjunct populations of P. alexiares in Mexico retain some ability to detoxify sweetbay, as do some individuals in most P. glaucus glaucus populations tested from ten different states north of Florida.     

Hybridization leads to host-use divergence in a polyphagous butterfly sibling species pair

Climate warming has lead to increased genetic introgression across a narrow hybrid zone separating the eastern and Canadian tiger swallowtails (Papilio glaucus and Papilio canadensis). This situation has led to the formation of an allochronically separated hybrid population with a delayed emerging phenotype or “late flight”. Here, we assess how the recombination of the parental genomes that lead to this phenotype may have facilitated another major ecological shift, host-use divergence. We first contrast the ovipositional profiles of the late flight population to that of the parental species P. glaucus and P. canadensis. Subsequently we contrast the larval survival and growth of the late flight, a P. canadensis and a P. glaucus population, and a population from the northern edge of the hybrid zone on five hosts. Our results indicate that the ovipositional preference of this hybrid swarm is identical to that of the introgressing parental species, P. glaucus. Due to the absence of the preferred hosts of P. glaucus (Liriodendron tulipifera L. and Ptelea trifoliata L.) where the late flight occurs, this ovipositional pattern implies a functional specialization onto a secondary host of both parental species, Fraxinus americana L. In contrast, the larval host-use abilities represent a mixture of P. glaucus and P. canadensis, indicating divergence in larval host-use abilities has not taken place. However, high genetic variability (genetic coefficient of variation) is present for growth on F. americana in the late flight hybrid swarm and tradeoffs for larval performance on the preferred hosts of the parental species are evident; indicating a strong potential for future specialization in larval host-use abilities. This current scenario represents an instance where a shift in a major ecological trait, host use, is likely occurring as a byproduct of a shift in an unrelated trait (delayed emergence) leading to partial reproductive isolation.     

Salicaceae detoxification abilities in Florida tiger swallowtail butterflies (Papilio glaucus maynardi Gauthier): Novel ability or Pleistocene holdover?

Abstract Florida populations of the eastern tiger swallowtail butterfly, Papilio glaucus L., have unique morphological features and ecological adaptations that have contributed to their subspecies status (P. g. maynardi Gauthier). We describe geographically unique abilities for detoxification of Carolina willow, Salix caroliniana Michx. (Salicaceae), for several Florida populations of P. g. maynardi. Of all the approximately 570 worldwide species of the Papilionidae, such Salicaceae detoxification abilities exist only in the allopatric North American western and northernmost species (P. rutulus Lucas, P. eurymedon Lucas and P. canadensis Rothschild & Jordan). Females of P. glaucus collected from populations in southeastern USA were examined for oviposition preference in 5‐choice assays, and displayed a low preference for Salicaceae (<5%), but larvae from Florida populations exhibited a high survival (>60%) on these plants. Detoxification abilities have previously shown to be autosomally inherited, and can be transferred via natural or hand‐paired interspecific hybrid introgression. However, these Florida populations are at least 700–1 500 km from the nearest hybrids or the hybrid species, P. appalachiensis Pavulaan & Wright, which possess these detoxification abilities. In any case, the Z ( = X)‐linked oviposition preferences for Salicaceae are lacking in these Florida populations, illustrating genetic independence of oviposition preference determination and larval survival/performance abilities. The origins of detoxification abilities are unlikely to be due to recent climate‐driven introgression, and may represent ancestral trait carry‐overs from interglacial refugium populations of the Pleistocene epoch.     

Effects of different leaf traits on growth rates of insect herbivores on willows

We examined relative effects of traits of leaf quality of ten willow species (Salix: Salicaceae) on growth rates of five species of insect herbivores found in interior Alaska (a willow sawfly, Nematus calais; the tiger swallowtail butterfly, Papilio canadensis; and three species of chrysomelid beetles, Gonioctena occidentalis, Calligrapha verrucosa, and Chrysomela falsa). Leaf traits examined were water content, toughness, total nitrogen contnet, pubescence, and presence or absence of phenolic glycosides. Of ten Salix species, four species contain phenolic glycosides in their leaves. We examined relative effects of water content, toughness, and nitrogen content of the Salix leaves on larval growth rates at three different levels, i.e., on a single host species, between different host species, and between herbivore species. The within-host analyses showed that effects of water content, toughness and/or nitrogen content on herbivore growth rates were generally significant in early-season herbivores but not in late-season herbivores. For each herbivore species, differences in growth rates between hosts were not explained by differences in water content, toughness, or nitrogen content. The between-herbivore analysis showed that the interspecific difference in larval growth rates were related to difference in water and nitrogen content of the hosts. Pubescence of Salix leaves had little effects on herbivore growth rates. Presence of phenolic glycosides had a positive effects on growth rates of a specialist, N. calais, but no effect on the other specialist, Ch. falsa. Presence of phenolic glycosides had, in general, negative effects on growth rates of nonspecialists, G. occidentalis, C. verrucosa, and P. canadensis.     

Larval developmental rates of three putative subspecies of tiger swallowtail butterflies,Papilio glaucus,and their hybrids in relation to temperature

Summary The eastern tiger swallowtail butterfly, Papilio glaucus L. has been presumed to be comprised of three parapatric subspecies (P. g. canadensis R & J, across Canada and the northern U.S.; P. g. glaucus L., in the eastern half of the U.S.; and P. g. australis Maynard, in Florida). Populations from 27° north latitude in Florida to 50° north latitude in Manitoba, Canada and from 75° west longitude in Pennsylvania to 96° west longitide in Canada were tested by laboratory larval feeding studies at various constant temperatures ranging from 16°–28° C. These data indicate that populations of Papilio glaucus show generally similar developmental responses to temperature, regardless of their geographic origin. Limited differences do exist between some populations studied, involving such traits as upper threshold temperature and differences between individual developmental rate curves. No consistent differences were noted between the three putative subspecies in their developmental response under these experimental conditions. In Wisconsin, the twenty year average of total season degree day accumulations above a base temperature (developmental threshold) of 10° C delineates a relatively narrow geographical region across the center of the state for which the bivoltine potential reaches its northernmost limits. This zone also closely corresponds to the southernmost limit of the P. g. canadensis subspecies (generally believed to be obligately univoltine).     

Biochemical ecology of the forest tent caterpillar: responses to dietary protein and phenolic glycosides

Summary Interactions between quaking aspen (Populus tremuloides) and the forest tent caterpillar (Malacosoma disstria) are likely to be influenced by leaf protein and phenolic glycoside levels, and insect detoxication activity. We investigated the direct and interactive effects of dietary protein and phenolic glycosides on larval performance and midgut enzyme activity of forest tent caterpillars. We conducted bioassays with six artificial diets, using both first and fourth stadium larvae. Four of the diets comprised a 2×2 factorial design-two levels of protein, each with and without phenolic glycosides. Additionally, we assayed high protein diets containing S,S,S-tributylphosphorotrithioate (DEF, an esterase inhibitor) and DEF plus phenolic glycosides. Enzyme solutions were prepared from midguts of sixth instars and assayed for -glucosidase, esterase and glutathione transferase activities. First instar mortality and development times were higher for larvae on diets low in protein or containing phenolic glycosides. Effects of phenolic glycosides were especially pronounced at low protein levels and when administered with DEF. Fourth instar development times were prolonged, and growth rates reduced, in response to consumption of low protein diets. Effects of phenolic glycosides on growth were less pronounced, although the effect for larvae on the low protein diet was nearly significant. Activity of each of the enzyme systems was reduced in larvae reared on low protein diets, and esterase activity was induced in larvae fed phenolic glycosides. Our results suggest that larval performance may be strongly affected by levels of protein and phenolic glycosides commonly occurring in aspen foliage, and that these factors may play a role in differential defoliation of aspen by forest tent caterpillars.     

Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes

Four esterase genes and general esterase activity were investigated in the gut of the termite Reticulitermes flavipes. Two genes (RfEst1 and RfEst2) share significant translated identity with a number of insect JH esterases. The two remaining genes (RfEst3 and RfEst4) apparently code for much shorter proteins with similarity to fungal phenolic acid esterases involved in hemicellulose solubilization. All four genes showed consistently high midgut expression. This result was further supported by colorimetric activity assays and Native polyacrylamide gel electrophoresis, which showed significant esterase activity and a number of isoforms in the midgut. The greatest esterase activity and isoform composition were detected when α‐naphthyl propionate was used as a substrate. Moreover, esterase activity and diverse isoforms were present in gut mitochondrial, microsomal, and cytosolic sub‐cellular protein fractions, as well as in the hindgut lumen. These findings reveal an agreement between gut esterase gene expression and activity distributions, and support the idea that R. flavipes gut esterase activity is host (not symbiont)‐derived. In addition, these findings support the hypotheses that termite gut esterases may play important roles in lignocellulose digestion and caste differentiation. This study provides important baseline data that will assist ongoing functional‐genomic efforts to identify novel genes with roles in semiochemical, hormone, and lignocellulose processing in the termite gut. © 2009 Wiley Periodicals, Inc.     

Extremely thermostable esterases from the thermoacidophilic euryarchaeon Picrophilus torridus

Two genes encoding esterases EstA and EstB of Picrophilus torridus were identified by the means of genome analysis and were subsequently cloned in Escherichia coli. PTO 0988, which is encoding EstA, consists of 579 bp, whereas PTO 1141, encoding EstB, is composed of 696 bp, corresponding to 192 aa and 231 aa, respectively. Sequence comparison revealed that both biocatalysts have low sequence identities (14 and 16%) compared to previously characterized enzymes. Detailed analysis suggests that EstA and EstB are the first esterases from thermoacidophiles not classified as members of the HSL family. Furthermore, the subunits with an apparent molecular mass of 22 and 27 kDa of the homotrimeric EstA and EstB, respectively, represent the smallest esterase subunits from thermophilic microorganisms reported to date. The recombinant esterases were purified by Ni²⁺ affinity chromatography, and the activity of the purified esterases was measured over a wide pH (pH 4.5–8.5) and temperature range (10–90°C). Highest activity of the esterases was measured at 70°C (EstA) and 55°C (EstB) with short pNP-esters as preferred substrates. In addition, esters of the non-steroidal anti-inflammatory drugs naproxen, ketoprofen, and ibuprofen are hydrolyzed by both EstA and EstB. Extreme thermostability was measured for both enzymes at temperatures as high as 90°C. The determined half-life (t _1/2) at 90°C was 21 and 10 h for EstA and EstB, respectively. Remarkable preservation of esterase activity in the presence of detergents, urea, and commonly used organic solvents complete the exceptional phenotype of EstA and EstB.     

Field bioassays show heterospecific mating preference asymmetry between hybridizing North American Papilio butterfly species (Lepidoptera: Papilionidae)

Few studies of interspecific mating preferences of naturally hybridizing species have been done in the field. Yet this is the only way potentially critical habitat-specific factors can be included in mating behavior evaluations. We conducted mate selection (male preference) studies using tethered pairs of heterospecific, size-matched virgin yellow females of Papilio glaucus and P. canadensis with free-flying male P. glaucus populations in south-central Florida, and with free-flying male P. canadensis populations in northern Michigan. Florida males clearly preferred the conspecific P. glaucus females of the pairs, as indicated by the touches/attempted matings and by the actual copulations observed. In 1997, 66.7% of the total attempts (n=168) and 94.2% of all copulations (n=69) were with the conspecific. In 1998 the same pattern of conspecific male preference was observed, with 67.3% of the touches/attempts (n=49) and 100% of the copulations (n=30) being with the P. glaucus females. In contrast, the natural populations of P. canadensis males did not show the expected conspecific preference. In fact, with wild P. canadensis males in 1997 a very strong preference was observed for the heterospecific P. glaucus females of the pairs, accounting for 75.8% of all touches/attempted matings (n=483) and 81.7% of all copulations (n=476). The strong asymmetry in interspecific mating preferences among these hybridizing Papilio species may reflect an ancestral trait in P. glaucus females that elicits strong preference in P. canadensis males, due perhaps to previously possessed male sensory bias or "good genes" indicators in the females. However, reinforcement of reproductive isolation in or near the hybrid zone of species overlap (sympatry) cannot be ruled out. Electronic Publication     

Chemical analysis of phenolic glycosides: art,facts, and artifacts

Summary Phenolic glycosides have been the subject of considerable interest in recent ecological and systematic studies, especially those involving the Salicaceae. But these compounds are markedly labile in aqueous media, and the consequences of spontaneous degradation for valid interpretation of results have been largely ignored by researchers. We found that freeze-drying and oven-drying of leaf samples from several Populus and Salix species produced dramatic changes in the total and relative concentrations of specific phenolic glycosides, when compared to analyses of fresh material. Extraction in aqueous and alcoholic media for extended (24 h) periods also effected changes in glycoside concentrations. Alterations in phenolic glycoside concentrations, interconversions among glycosides, and production of artifactual glycosides result from a series of hydrolytic reactions. These deleterious effects can be best (but not entirely) avoided by the use of fresh plant material, cold, nonaqueous extraction solvents, and short extraction times. Because individual phenolic glycosides exhibit very different biological activities against herbivores, we caution ecologists to use utmost care in the performance and interpretation of phenolic glycoside assays.     

Small enzymes with esterase activities from two thermophilic fungi, Emericella nidulans and Talaromyces emersonii

Extracellular esterase activities in Emericella nidulans and Talaromyces emersonii are attributed to small enzymes with molecular weights less than 10 kDa (microenzymes). A 1.6 kDa esterase accounted for most of the esterase activity observed in both organisms and one of them also contained a 4.1 kDa microenzyme with weaker esterase activity. These esterases were growth-associated and active towards fluorescein dibutyrate and -naphthyl acetate as well as tributyrin.     

INSECTICIDE RESISTANCE IN THE GROUND SPIDER,Pardosa sumatrana (THORELL, 1890; ARANEAE: LYCOSIDAE)

Elevated levels of insecticides detoxifying enzymes, such as esterases, glutathione S‐transferases (GSTs), and cytochrome P‐450 monooxygenases, act in the resistance mechanisms in insects. In the present study, levels of these enzymes in the insecticide‐resistant ground spider Pardosa sumatrana (Thorell, 1890) were compared with a susceptible population (control) of the same species. Standard protocols were used for biochemical estimation of enzymes. The results showed significantly higher levels of nonspecific esterases and monooxygenases in resistant spiders compared to controls. The activity of GSTs was lower in the resistant spiders. Elevated levels of nonspecific esterases and monooxygenases suggest their role in metabolic resistance in P. sumatrana. The reduced levels of total protein contents revealed its possible consumption to meet energy demands.     

GENETICS OF MIMICRY IN THE TIGER SWALLOWTAIL BUTTERFLIES,PAPILIO GLAUCUS AND P. CANADENSIS (LEPIDOPTERA: PAPILIONIDAE)

The tiger swallowtail butterfly, Papilio glaucus, exhibits a female-limited polymorphism for Batesian mimicry; the Canadian tiger swallowtail, Papilio canadensis, lacks the mimetic (dark) form entirely. The species hybridize to a limited extent where their ranges overlap. Field collections and censuses indicate that mimetic females occur throughout the range of P. glaucus but at lowest frequencies in populations at the latitudinal edges of its geographic range such as the southernmost part of Florida and along the entire northern edge of its distribution from Massachusetts to Minnesota. Frequencies of mimetic females have remained relatively stable over time. Inheritance of the mimetic form is controlled primarily by two interacting sex-linked loci. The typical matrilineal pattern of inheritance in P. glaucus can be explained by polymorphism at a Y-linked locus, b. Analysis of P. glaucus × P. canadensis crosses has also revealed an X-linked locus, s, which controls the expression of the mimetic phenotype. The P. canadensis allele, s^can, suppresses the mimetic phenotype in hybrid and backcross females. Results from more than 12 yr of rearing tiger swallowtails, including interspecies hybrids, indicate that the absence of mimetic P. canadensis females is due to both a high frequency of the “suppressing” allele s^can and low frequency of the black-pigment-determining b + allele. The frequency of s^can (or other suppressing alleles of s) in P. glaucus populations outside the hybrid zone is low. Some males heterozygous at the s locus and some suppressed mimetic females occur within the hybrid zone. A simple genetic model predicts the frequency of daughters that differ in phenotype from their mothers.     

Esterases in pollen and stigma of Brassica

The dry type stigma of Brassica is covered with a continuous layer of cuticle. Cutinase and non-specific esterases may be involved in breakdown of this cuticle barrier during pollen-stigma interaction, but only a little is known about their nature and characteristics. We report here the presence of two distinct esterases from stigma and pollen of Brassica. A 33 kD esterase assayed using MU-butyrate substrate shows high activity in stigma papillae. A similar esterase from Tropaeolum pollen has been shown to possess active cutinase activity. The esterase activity in anther tissue is due to a 24 kD enzyme with substrate specificity toward acetate esters. Both enzymes require sulfhydryl groups for their catalytic activity. Immunogold labelling of antibodies raised against these esterases localised the proteins at the subcellular level. Antibodies for MU-butyrate hydrolase gave a positive signal in the cell walls of mature stigma papillae and in the tapetum and microspores during early stages of anther development. In the mature anther, a positive signal in the cytoplasm of pollen grains with some detectable localisation in the exine layer of the pollen wall was obtained. Similar results were obtained with acetate hydrolase antibodies. These esterases are thus spatially and temporally regulated in stigma and anther tissues.Abbreviations MU methyl umbelliferyl - pAbC anti-butyrate hydrolase polyclonal antibodies - pAbE anti-acetate hydrolase polyclonal antibodies     

Willow genotype, but not drought treatment, affects foliar phenolic concentrations and leaf-beetle resistance

In a greenhouse experiment we examined the effect of willow genotype and irrigation regime (moderate drought and well‐watered) on plant growth parameters, foliar nitrogen, and phenolic concentrations, as well as on the preference and performance of the blue leaf beetle, Phratora vulgatissima (L.) (Coleoptera: Chrysomelidae). The 10 vegetatively propagated willow genotypes in the experiments were F2 full‐sibling hybrids, originated from a cross between Salix viminalis (L.) (Salicaceae) (high in condensed tannins) and Salix dasyclados (L.) (Salicaceae) (rich in phenolic glycosides). Insect bioassays were conducted on detached leaves in Petri dishes as well as with free‐living insects on intact potted plants. The 10‐week long irrigation treatments caused statistically significant phenotypic differences in the potted willow saplings. Total biomass was somewhat higher in the well‐watered treatment. The root to total biomass ratio was higher in the drought‐treatment plants. There was significant genotypic variation in foliar nitrogen concentrations, and they were higher in the drought‐treatment plants. There was also a strong genotypic variation in each of the phenolic substances analyzed. Condensed tannins, which accounted for the greatest proportion of total phenolic mass, were higher in the well‐watered treatment. There was, however, no difference in levels of the other phenolics (salicylates, cinnamic acid, flavonoids, and chlorogenic acid) between irrigation treatments. The sum of these phenolics was higher in the well‐watered treatment. There was a strong variation in P. vulgatissima larval development on different willow genotypes, and larval performance was negatively correlated with levels of salicylates and cinnamic acid. There was, however, no effect of irrigation treatment on larval performance. Phratora vulgatissima preferred to feed on well‐watered plants, and we found a preference for oviposition there, but neither feeding nor oviposition site preference was affected by willow genotype. Adult feeding and oviposition preferences were not correlated with larval performance.     

Two glucuronoyl esterases of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The white-rot fungus Phanerochaete chrysosporium produces glucuronoyl esterase, a recently discovered carbohydrate esterase, during growth on sugar beet pulp. Two putative genes encoding this enzyme, ge1 and ge2, were isolated and cloned. Heterologous expression in Aspergillus vadensis, Pycnoporus cinnabarinus and Schizophyllum commune resulted in extracellular glucuronoyl esterase activity, demonstrating that these genes encode this enzymatic function. The amino acid sequence of GE1 was used to identify homologous genes in the genomes of twenty-four fungi. Approximately half of the genomes, both from ascomycetes and basidiomycetes, contained putative orthologues, but their presence could not be assigned to any of fungal class or subclass. Comparison of the amino acid sequences of identified and putative glucuronoyl esterases to other types of carbohydrate esterases (CE) confirmed that they form a separate family of CEs. These enzymes are interesting candidates for biotechnological applications such as the separation of lignin and hemicellulose.