Correlation Between the Mesodermal Male Genital Ducts and the Spermatophore Structure in a Ditrysian Moth,Zygaena trifolii (Esper, 1783) (Insecta,Lepidoptera, Zygaenidae)

Abstract The mesodermal male genital system and the spermatophore of Zygaena trifolii (Insecta, Lepidoptera, Zygaenidae) have been studied histologically. Whereas the long unpaired duct, the ductus mesospermaticus, has been found to consist of at least six distinctly different regions, the tissues and secretions of the seminal vesicles and the accessory glands were found to be quite uniform. Several of the different regions are separated from each other by epithelial septa (constrictions). The external structure and the internal organization of the spermatophore are described. Based on histological comparisons, the correlation between the contents of the spermatophore and the different parts of the male system is given.     

Genetic variability in the diapause response of the burnet moth Zygaena trifolii (Lepidoptera: Zygaenidae)

A high degree of phenotypic variability was observed in the diapause response of the burnet moth Zygaena trifolii. In this study, we show that the observed variability is partly based on genetic differences between individuals. In a selection experiment, the larval instar at which diapause occurs was changed within six generations. Diapause instars were dependent on the time of pre-diapause development of larvae, which varied considerably between larvae. A heritability analysis indicates that a part of the variability in development time is based on additive genetic variance. The maintenance of genetic variability in the development time and the diapause response of Z. trifolii is discussed in the context of spatially and temporally changing selection pressures.     

Repeated larval diapause and diapause-free development in geographic strains of the burnet moth Zygaena trifolii Esp. (Insecta,Lepidoptera)

Summary Zygaena trifolii is a long-day insect with temperature-dependent photoperiodic responses. All larval instars are sensitive to photoperiod; however, diapause may occur at the third larval stage or any subsequent larval instars. There were quantitative differences within populations in the threshold photoperiod for diapause induction. The diapause response was polymorphic, so that larvae might enter diapause at different instars under the same culture conditions. Furthermore, decreasing photoperiods below a critical daylength shifted the diapausing instar towards earlier stages. Geographic strains of Z. trifolii showed discontinuous clinal variation. Near the northern edge of the distribution [Cologne (Köln), FRG], there is first an obligatory diapause, mainly during early instars, and additional facultative (repeat) diapauses during later larval instars in subsequent years. In the southern part of its distribution, this burnet moth is partially bivoltine in the field with a facultative first developmental arrest and a decreased capacity for repeated diapause (Valencia, Spain; Marseille, France). Further experiments indicated that the photoperiodically controlled diapause reaction is also influenced by the number of photoperiodic cycles experienced during the period spent in each larval instar, which depends on temperature. The adaptive significance of obligatory and facultative repeated diapause, varying even among the offspring of a single female, may be to buffer the populations against the more extreme and, from year to year, unpredictable fluctuations in climatic conditions at the northern edge of the distribution.Abbreviations L₃ feeding 3rd larval instar - L₄D diapausing 4th larval instar - L₅D2 repeat-diapausing larval instar with second diapause at the 5th larval stage - LD light-dark cycle - KT shortday conditions (e.g. LD 8:16) - LT long-day conditions (e.g. LD 16:8)     

Little-known accessory glands in female Zygaena moths (Lepidoptera, Zygaenidae)

In the female genital system of Zygaena moths, an additional pair of accessory glands is present besides the Y-shaped sebaceous gland. The term 'Petersen's glands' is proposed for these organs. Anatomy, histology, histochemistry and cytology of Petersen's glands of Zygaena trifolii are described. The sac-like glands, situated in the extreme dorsocaudal part of the abdomen, can be divided into a purely secretory part consisting of acini with large pear-shaped gland cells and a reservoir part with combined secretory and storage function. The secretory cells of the acini are penetrated by long curved ductules or secretory end apparatuses having feltwork consisting of very fine filaments. The cytoplasm is characterized by abundance of smooth tubular endoplasmic reticulum (ATER) and the presence of peroxisomes. This cytoplasmic organization is in accordance with the chemical composition of the sticky secretion, which evidently consists completely of lipids. The ultrastructure of the epithelium lining the reservoir of the glands has both traits of secretory and of transporting epithelia. Besides contributing to the secretion, it may be involved in absorption of residual aqueous phase from the contents of the reservoir.     

Cyanoglucoside storing cuticle of Zygaena Larvae (Insecta,Lepidoptera)

Summary As a deterrent against predators, larvae of Zygaena trifolii release droplets of fluid containing cyanoglucosides from segmentally arranged cuticular cavities. Histological examinations show that during the moulting period, the old cuticle, including the cavities and the secretion within them, is degraded, with the exception of a thin mesocuticular layer forming the exuviae. When the endocuticular layer of the new cuticle is deposited, the cuticle detaches from the underlying epidermis in specific areas, which leads to the formation of the cuticular cavities. During a moult-intermoult sequence the concentration of cyanoglucosides in both the haemolymph and the defensive secretion shows specific changes. These changes seem to be related to the formation and degradation of the cavities. We suggest that during the moult the cyanoglucosides are transported through the epidermis into the haemolymph to prevent them from being wasted with the exuviae and, after ecdysis, are retranslocated into the newly formed cavities.     

Allozymic and morphometric analysis of populations in the Zygaena purpuralis complex (Lepidoptera, Zygaenidae)

In Central Europe and Asia Minor Zygaena purpuralis and Z. minos are two sibling species exhibiting a considerable geographic variation in several features. In this study we examine some populations from the Central and Southern Apennines (Italy) by comparing multivariate analyses of individual allozymic multilocus profiles and of individual morphometric profiles of male genitalia in order to assess their genetic and phenetic relationships.
Results indicate that populations of Z. purpuralis in the Apennines behave in rather different ways with respect to the occurrence of gene exchange with other gene pools. No gene flow appears to occur between two geographically close southern populations; however, extensive introgression, revealable at both the allozymic and morphometric level, seems to occur between some purpuralis and minos -like sympatric populations from Abruzzi. Evolutionary relationships among these peninsular Italy populations are discussed with reference to historical factors, that is, the occurrence of multiple Pleistocene refugia, which could have determined different degrees of isolation among gene pools. This situation is rather different from that previously outlined for Central Europe and present results suggest that the Z. purpuralis complex might include more than two biological species.     

Intimate roles for cyanogenic glucosides in the life cycle of Zygaena filipendulae (Lepidoptera, Zygaenidae)

Zygaena larvae sequester the cyanogenic glucosides (CNglcs) linamarin and lotaustralin from their food plants (Fabaceae) and also de novo biosynthesize these compounds. In Zygaenidae, CNglcs serve as defence compounds during the entire life cycle, and their content and ratio are tightly regulated. We demonstrate that Z. filipendulae males transfer a nuptial gift of CNglcs to females during mating, and that females prefer males with a higher content of CNglcs for mating. Average HCN emission from female imagines is 19 times higher than from males, suggesting that plumes of HCN emitted from the perching female may serve to attract flying males. Analysis of the linamarin and lotaustralin content and ratio within different tissues in Z. filipendulae larvae shows that integument and haemolymph constitute the main sites of CNglc deposition. The data suggest that CNglcs may serve an additional role as storage compounds of reduced nitrogen that is mobilized during the transition of the last instar larva to imago, most likely to provide nitrogen for chitin synthesis. At least one of the enzymes responsible for de novo biosynthesis of CNglcs in Z. filipendulae is located in the integument. In conclusion, CNglcs play many important and different roles during the entire life cycle of Z. filipendulae in addition to defence.     

Molecular biogeography of the arctic-alpine disjunct burnet moth species Zygaena exulans (Zygaenidae, Lepidoptera) in the Pyrenees and Alps

Aim The phylogeography of ‘southern’ species is relatively well studied in Europe. However, there are few data about ‘northern’ species, and so we studied the population genetic structure of the arctic‐alpine distributed burnet moth Zygaena exulans as an exemplar. Location and methods The allozymes of 209 individuals from seven populations (two from the Pyrenees, five from the Alps) were studied by electrophoresis. Results All 15 analysed loci were polymorphic. The mean genetic diversities were moderately high (A: 1.99; H_e: 11.5; P: 65%). Mean genetic diversities were significantly higher in the Alps than in the Pyrenees in all cases. F_ST was 5.4% and F_IS was 10%. Genetic distances were generally low with a mean of 0.022 between large populations. About 62% of the variance between populations was between the Alps and the Pyrenees. The two samples from the Pyrenees had no significant differentiation, whereas significant differentiation was detected between the populations from the Alps (F_ST = 2.8%, P = 0.02). Main conclusion Zygaena exulans had a continuous distribution between the Alps and the Pyrenees during the last ice age. Most probably, the species was not present in Iberia, and the samples from the Pyrenees are derived from the southern edge of the glacial distribution area and thus became genetically impoverished. Post‐glacial isolation in Alps and Pyrenees has resulted in a weak genetic differentiation between these two disjunct high mountain systems.     

Ultrastructure of the larval corpus allatum of Hyphantria cunea drury (Insecta,Lepidoptera)

Summary The corpora allata of the three last larval instars were studied in newly molted animals, at the beginning, middle, and end of the feeding period, and during the molt period. They were found to consist of uniform gland cells, whose ultrastructure changes in the course of the instars.In gland cells considered to be resting, the outer and inner nuclear membranes run in parallel without forming a dilated perinuclear space. Mitochondria are small, polymorphic, with an electron-dense matrix. The smooth endoplasmic reticulum (SER) appears as stacks of parallel cisternae near the nuclear envelope and in the rest of the cytoplasm, and as accumulations of twisted profiles. Occasionally, the SER takes the form of paracrystalline bodies. There are few small smooth-surfaced vesicles in the cytoplasm.In cells considered as active, a dilated perinuclear space occurs. The peripheral ends of profiles forming the SER are swollen, and numerous vesicles and vacuoles bud off from them to fill the cytoplasm. Mitochondria are large, with a more transparent matrix. The plasma membrane of gland cells located just beneath the connective tissue sheath forms numerous small invaginations.The corpora allata consist of resting cells during the molt periods. At the beginning of each instar, few active gland cells appear. In the middle of the second to last and the third to last instars, the bulk of the gland cells is active. At the end of these instars, there are both active and inactive cells. In the middle of the last instar, the gland cells are inactive or subactive, and at its end, all gland cells are completely inactive.     

The internal female genitalia of some Zygaenidae (Insecta,Lepidoptera): their morphology and remarks on their phylogenetie significance*

The internal female genitalia of fifteen species of Zygaenidae (representing seven genera of Zygaeninae), one species of Heterogynidae and one species of Limacodidae have been studied in order to delimit the distribution of Petersen's glands within the Zygaenidae. The study demonstrates a considerable amount of structural variation. Petersen's glands are found to represent an autapomorphic character of the Zygaeninae, while the pseudobursa is restricted to the Zygaenini. Most likely it is an autapomorphic character of this tribe. Some other characters that were studied are discussed with special reference to their usefulness for a reconstruction of the phylogeny of the Zygaenidae.     

Virulence of Candida albicans mutants toward larval Galleria mellonella (Insecta, Lepidoptera, Galleridae)

Culture medium affected the virulence of a strain of Candida albicans toward Galleria mellonella larvae, but the yeast growth rates in yeast extract - peptone - dextrose broth and synthetic Galleria serum were not correlated with yeast virulence. Virulent C. albicans grew rapidly in larval serum, whereas, it limited nodulation and continued development in vivo, producing toxins that damaged the hemocytes and fat body. Nonpathogenic yeast-phase cells grew slowly in larval serum but induced extensively melanized nodules in vivo and developed no further. There was no discernible relationship in 14 exo-enzymes between the virulent and avirulent yeast strains and virulence. The avirulent myosin-I-defective yeast cells were rapidly removed from the hemolymph in vivo because of lysozyme-mediated yeast agglutination and the possible binding of the yeast cells by lysozyme and apolipophorin-III. Both lysozyme and apolipophorin-III are proteins that bind beta-1,3-glucan. Finally, insects with nonpathogenic C. albicans exhibited induced immunity and were more resistant to candidiasis from the wild-type yeast cells than were noninduced insects.     

The cyanogenic glucoside composition of Zygaena filipendulae (Lepidoptera: Zygaenidae) as effected by feeding on wild-type and transgenic lotus populations with variable cyanogenic glucoside profiles

Zygaena larvae sequester the cyanogenic glucosides linamarin and lotaustralin from their food plants (Fabaceae) as well as carry out de novo biosynthesis of these compounds. In this study, Zygaena filipendulae were reared on wild-type Lotus corniculatus and wild-type and transgenic L. japonicus plants with differing content and ratios of the cyanogenic glucosides linamarin and lotaustralin and of the cyanoalkenyl glucosides rhodiocyanoside A and D. LC-MS analyses, free choice feeding experiments and developmental studies were used to examine the effect of varying content and ratios of these secondary metabolites on the feeding preferences, growth and development of Z. filipendulae. Larvae reared on cyanogenic L. corniculatus developed faster compared to larvae reared on L. japonicus although free choice feeding trials demonstrated that the latter plant source was the preferred food plant. Larvae reared on acyanogenic L. corniculatus showed decelerated development. Analysis of different life stages and tissues demonstrate that Z. filipendulae strive to maintain certain threshold content and ratios of cyanogenic glucosides regardless of the composition of the food plants. Despite this, the ratios of cyanogenic glucosides in Z. filipendulae remain partly affected by the ratio of the food plant due to the high proportion of sequestering that takes place.     

Characterization of microsatellite loci for Reissita simonyi (Rebel, 1899) (Lepidoptera,Zygaenidae)

The newly developed microsatellite set for Reissita simonyi ( Rebel 1899 ) (Lepidoptera, Zyganidae) is presented. In total, 14 loci turned out to be highly polymorphic. The microsatellite set will be used for a population genetic analysis of this endemic species in Southern Arabia. In the focus of this study stands the genetic differentiation and variability of isolated populations, which are assumed to be based on habitat fragmentation due to natural processes and human impact. Moreover, this new microsatellite set showed accurate band patterns in the Palaearctic genus Zygaena which seems to be promising for further studies.     

Remarks on the family-level phylogeny of butterflies (Insecta, Lepidoptera, Rhopalocera)

1. Available evidence on butterfly family-level relationships is re-examined according to the principles of phylogenetic (cladistic) systematics. 2. The assumption of a sister-group relationship between the Hesperioidea and Papilionoidea seems a reasonably substantiated working hypothesis. 3. The Papilionoid families Papilionidae, Pieridae and Lycaenidae sensu Ehrlich (1958) are definable as monophyletic entities; of Ehrlich 's two remaining families, Nymphalidae and Libytheidae, the former is paraphyletic in terms of the latter. 4. The interrelationships between the Papilionoid families may be presented as Papilionidae + (Pieridae + [Lycanidae + Nymphalidae]). 5. In a phylogenetic system any given arrangement of taxa is either correct or not: Contrary to the pheneticists' view (Ehrlich and Ehrlich 1967) phylogenetic systematists cannot accept the existence of a multitude of valid classifications.     

Bacterial formyl peptides affect the innate cellular antimicrobial responses of larval Galleria mellonella (Insecta: Lepidoptera)

The non-self cellular (hemocytic) responses of Galleria mellonella larvae, including the attachment to slides and the removal of the bacteria Xenorhabdus nematophila and Bacillus subtilis from the hemolymph, were affected by N-formyl peptides. Both N-formyl methionyl-leucyl-phenylalanine (fMLF) and the ester derivative decreased hemocyte adhesion in vitro, and both elevated hemocyte counts and suppressed the removal of both X. nematophila and B. subtilis from the hemolymph in vivo. The amide derivative and the antagonist tertiary-butoxy-carbonyl-methionyl-leucyl-phenylalanine (tBOC) increased hemocyte attachment to glass. The fMLF suppressed protein discharge from monolayers of granular cells with and without bacterial stimulation, while tBOC stimulated protein discharge. The peptide tBOC offset the effects of fMLF in vitro and in vivo. This is the first report implying the existence of formyl peptide receptors on insect hemocytes in which the compounds fMLF and tBOC inhibited and activated hemocyte activity, respectively.     

The larval abdomen of the enigmatic Nannochoristidae (Mecoptera,Insecta)

External and internal structures of the larval abdomen of Nannochorista are described in detail, with emphasis on the posterior segments. The results are compared with conditions found in other groups of Antliophora, especially the mecopteran subgroups Boreidae and Pistillifera. Like the entire postcephalic body, the larval abdomen of Nannochorista is extremely slender and nearly cylindrical. The anterior segments are largely unmodified. The surface is smooth and lacks any protuberances or prolegs. The term “cloaca” for the posterior membranous pouch of Nannochorista sp. is morphologically unjustified. A list of muscles of segments IX and X is presented. The abdominal musculature was partly homologized following Snodgrass. The muscles of segment X are highly modified. They move the membranous pouch, the anal papillae, and the terminal lobes. The presence of these structures is likely an adaptation to the specific aquatic life style of nannochoristid larvae. The anal papillae are possibly homologous to the 4-lobed terminal attachment apparatus of larvae of Caurinus (Boreidae) and Pistillifera (Panorpidae, Bittacidae, Choristidae) but this is uncertain. The specific condition in both groups, i.e. two retractile papillae with tracheae and Malpighian tubules in Nannochoristidae, and a 4-lobed exposed attachment device in Pistillifera + Boreidae (groundplan) are very likely autapomorphic for both groups, respectively. A slender abdomen with smooth surface is very likely plesiomorphic within Antliophora and Mecopterida. This condition is found in Trichoptera (partim), Nannochoristidae, Siphonaptera, and many basal groups of Diptera. An eruciform or scarabaeiform body shape with a soft, largely unsclerotised cuticle is probably a synapomorphy of Boreidae and Pistillifera. The presence of ventral protuberances resembling prolegs on the anterior segments is an autapomorphy of the latter group. The homology of paired or unpaired terminal appendages of segment X is uncertain. However, the specific condition of paired and 3-segmented appendages with hooks in Nannochoristidae is almost certainly autapomorphic for this family. The protracted opening of the hind gut on the membranous pouch is another potential autapomorphy of Nannochoristidae. Aquatic habits of larvae, also very likely an apomorphic condition, have likely evolved several times independently in Antliophora.     

The proboscis of eye-frequenting and piercing Lepidoptera (Insecta)

The external structures of the proboscis are investigated in eye-frequenting species of Noctuidae, Geometridae and Pyralidae by means of scanning electron microscopy. They are compared with non-eye-frequenting representatives of these families. In Noctuidae, highly specialized fruit-piercing, skin-piercing blood-sucking, and sweat-feeding representatives have been included. All hemi- and eulachryphagous species have a soft proboscis tip which is characterized by few sensilla and strongly elongated, dentate plates of the dorsal galeal linkage. The latter structures leave broad gaps between them that lead into the food canal at the tip. This arrangement permits the uptake of fluid suspensions such as lachrymal fluid, wound exudates and pus. The modified dorsal galeal linkage is regarded as an adaptation for this highly derived feeding habit. The rough surface of the proboscis is likely to cause irritation and possible mechanical damage to the conjunctiva and cornea which results in an increased lachrymal flow and production of pus. In contrast to fruit-piercing and skin-piercing Noctuidae, there are no erectile structures on the proboscis of eye-frequenting species.—The comparison with related non-eye-frequenting species demonstrates that the particular morphology of the proboscis tip in lachryphagous moths evolved convergently in different families of Leipdoptera.     

Immunolocalization of odorant-binding proteins in noctuid moths (Insecta, Lepidoptera)

Odorant-binding proteins were studied in the noctuid moths Agrotis segetum, Autographa gamma, Helicoverpa armigera, Heliothis virescens and Spodoptera littoralis using antisera raised against the pheromone-binding protein (PBP) and general odorant-binding protein 2 (GOBP2) of Antheraea polyphemus (Saturniidae). Proteins immunoreacting with these antisera were only found on the antennae and PBP and GOBP2 could be identified on western blots of males and females of all five species. PBPs were predominantly localized in sensilla trichodea and GOBP2 in sensilla basiconica, in good correlation with the stimulus specificity of the receptor cells in these sensilla. In H. armigera and H. virescens the majority of the s. trichodea immunoreacted with the antiserum against PBP of A. polyphemus; in A. segetum, A. gamma and S. littoralis, on the other hand, a high percentage of s. trichodea remained unlabelled. Probably, the PBP expressed in these sensilla is so different that it does not immunoreact with the antiserum used. Such a protein was found by native PAGE of antennal extracts of A. segetum and S. littoralis. These data correlate with the fact that the two heliothine species use pheromones with the same alkyl chain length as A. polyphemus, while the other three species use pheromones with shorter chains. In H. armigera, H. virescens, A. gamma and S. littoralis female antennae were also immunolabelled and a large number of PBP-expressing s. trichodea was consistently found. In S.littoralis this fits with the electrophysiologically recorded high pheromone sensitivity of female s. trichodea, whereas in females of H. armigera and H. virescens no or only weak responses to pheromone stimulation have been reported. Therefore, PBP expression in a sensillum does not necessarily imply pheromone sensitivity of its receptor cells.     

The adhesive devices in larvae of Lepidoptera (Insecta, Pterygota)

Adhesion to smooth surfaces by means of thin fluid lipid film was studied on living larvae of 71 species of Lepidoptera by a simple ”light reflection method”. The method made it possible to localize exactly the sites of adhesion and to estimate roughly the film thickness, within a certain range. Furthermore, it revealed the general presence of mobile lipid on the entire insect surface. The observations on living larvae were complemented by comparative structural studies of the adhesive parts with light and scanning electron microscopes on preserved specimens of 161 species. Specialized adhesive devices were found in great diversity on larval legs and prolegs, especially in larvae living in the open air on their food plants. Two main surface types of adhesive cuticle were found: (1) cuticle with a flexible smooth surface and (2) cuticle with very numerous small projections (microtrichia) with spatulate and recurved apices. Both the functional implications of the adhesive cuticular structure and the role of the adhesive fluid as well as the evolution of the adhesive devices are discussed. The adhesive effect is due to ”capillary” or meniscus forces. Accepted: 29 July 1999     

Structural mutants of the W chromosome in Ephestia (Insecta,Lepidoptera)

In a search for genetic markers of W-chromosome-autosome fusions in Ephestia, two closely linked autosomal markers, ml and Us, were found to show sex linkage in several families of chromosome mutant strains. In these families, the wild-type allelomorphs, ml ⁺ and US ⁺, label the autosome that is translocated to the W chromosome. With ml (musterlos) a sex dimorphic strain could be established in which males (ml/ml) have patternless wings and females (ml ⁺/ml) have the normal wing pattern.—Using these genetic markers, stability of the fusion chromosome was studied. Recurrence to autosomal inheritance of the marker occurs at a considerable rate. In two chromosome fusion strains, a cytogenetically detectable breakage of the fusion giving rise to a wild-type-like W chromosome was the predominant cause for the recurrence of the marker to autosomal inheritance. In a third strain a more complicated chromosome rearrangement was the predominant cause: the translocated autosome was replaced by a non-homologous one, presumably after a cytogenetically undetectable breakage event of the original fusion. — The high rate of breakage suggests that the fusion chromosomes are dicentrics, a situation not compatible with a typical holokinetic organization of Lepidoptera chromosomes.