Isolation and identification of 9-methylgermacrene-B as the putative sex pheromone of Lutzomyia cruzi (Mangabeira, 1938) (Diptera: Psychodidae)

Lutzomyia (Lutzomyia) cruzi has been named as a probable vector of Leishmania chagasi in Corumbá, Mato Grosso do Sul, Brazil. Taxonomically L. cruzi is closely related to the L. longipalpis species complex. Females of L. cruzi and L. longipalpis are morphologically indistinguishable and associated males must be examined carefully to confirm identifications. Chemical analysis hexane extracts of male L. cruzi has revealed the presence of a 9-methylgermacrene-B (C16), a homosesquiterpene (mw 218) previously shown to be the sex pheromone of one of the members of the L. longipalpis species complex.     

Genetic differentiation in natural populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae) with different phenotypic spot patterns on tergites in males

Entomological surveys in the state of Maranh?o have recorded morphologically distinct populations of Lutzomyia longipalpis (Lutz & Neiva). Some populations have one pair of spots (1S) on the fourth tergite, while others have two pairs (2S) on the third and fourth tergites of males. In the present study we investigated the degree of genetic polymorphism among four populations in the municipalities of Caxias, Codó and Raposa, in the state of Maranh?o, Brazil, by using RAPD (Random Amplified Polymorphic DNA) markers. A total of 35 loci were identified, of which 30 were polymorphic. The highest polymorphism was observed with primer OPA 4, which produced 11 different profiles. Genetic diversity was assessed using grouping methods that produced a dendrogram in which the genotypes could be clearly separated into two main clades according to the number of spots on the male abdominal tergites. One cluster contained the populations from Caxias and Codó, and the other was formed by the populations from Raposa and Codó. The results of our RAPD analysis showed a clear separation between the populations with one and two pairs of spots. The epidemiologic significance of this genetic differentiation should be investigated in future studies.     

Effects of the juvenile hormone analog pyriproxyfen on German cockroach, Blattella germanica (L.), tergal gland development and production of tergal gland secretion proteins

Male German cockroaches possess secretory glands that secrete fluid into a pair of transverse depressions on the seventh and eighth abdominal tergites. We investigated the effects of altered juvenoid titer during the first part of the last instar on tergal gland secretory tissue development and the production of tergal gland secretion proteins. Male fifth (last) instar nymphs (1-3 days post-emergent) were topically treated with the JH analog pyriproxyfen. Light and transmission electron microscopy demonstrated that treatment with pyriproxyfen produced a visible decrease in the amount of tergal gland tissue present, a deformation of the overall shape of the gland located on tergite seven, and a less orderly arrangement of the secretory cells in the tissue. The protein fraction of tergal gland secretion was examined in pyriproxyfen-treated and control insects 1, 5, and 15 days after the insects molted to the adult stage. Amounts of all tergal secretion proteins were reduced in treated insects.     

Terga1 and inter–tergal metasomal glands of male braconine wasps (Insecta, Hymenoptera, Braconidae)

The distribution of cxocrine tergal and inter–tergal glands among males of more than sixty genera of Braconinac has been surveyed. The morphology of the glands and their reservoirs is described. Two gland types were found. Large, sac–like inter–tergal glands, usually most prominent between the 7th and 8th abdominal tergites, were found in all members of the Braconini, Adeshini, Aphrastobraconini, Glyptomorphini and the Atanycolus and Merinotus groups of genera. Inter–tergal glands were generally considerably smaller in members of the Bathyaulacini, Coeloidini. Euurobraconini and Iphiaulacini. Small pore–plate, vase–like or tubular tergal glands opening on the medio–lateral or antero–lateral selerotized parts of the 8th metasomal tergite were found in many genera, but were largely absent from members of the Braconini, Euurobraconini and Iphiaulacini ss. The phylogenetic implications of these findings are discussed.     

The effect of male sandfly pheromone and host factors as attractants for female Lutzomyia longipalpis (Diptera: Psychodidae)

Abstract Male Lutzomyia longipalpis (Lutz & Neiva) pheromone extracted from tergal glands combined with heat, carbon dioxide gas and hamster urine was found to be attractive to virgin female sandflies. The host factors if offered in the absence of pheromone or a heat source were, however, unattractive to female flies. Pheromone stored for 6 days and then combined with the same host components remained attractive to female flies but storage failed to enhance its attractiveness. The effects of carbon dioxide gas, hamster urine, chicken uropygial gland extract and octenol plus acetone on the responses of females were inconsistent. A ceramic battery powered heat source was tested with pheromone and host attractants and showed potential for field application in developing a pheromone trap.     

Life cycle differences among Brazilian sandflies of the Lutzomyia longipalpis sibling species complex

The developmental cycles of five Brazilian populations of the Lutzomyia longipalpis Lutz & Neiva species complex (Diptera: Psychodidae) were compared under laboratory conditions. Three of the populations were derived from insects collected in allopatric sites at Natal (Rio Grande do Norte State), Jacobina (Bahia State) and Lapinha Cave (Minas Gerais State). The other two originated from Sobral (Ceará State), where the males of two sympatric species can be distinguished by the presence of one (1S) or two (2S) pairs of abdominal spots. The results of the present study clearly show that all three populations whose males produce C₁₆ pheromones and use pulse-type copulation songs (Jacobina, Lapinha Cave and Sobral 1S) are more easily adapted to the colonization conditions used in our laboratory, producing larger egg batches, with higher survival and an overall faster developmental cycle. This contrasts with populations producing C₂₀ male pheromones and using burst-type copulation songs (Natal and Sobral 2S) that produce smaller egg batches, have higher oviposition mortality and a slower rate of development under identical laboratory conditions. In conclusion, these phenological differences are a further indication of the differentiation of the siblings within the Lu. longipalpis species complex.     

Structure and evolution of stridulatory mechanisms in New Zealand wetas (Orthoptera : Stenopelmatidae)

Four types of stridulatory mechanisms were found in 35 species (7 genera) of New Zealand wetas (Orthoptera : Stenopelmatidae). These include: (1) tergo-femoral; (2) tergo-tergal; (3) mandbbuao-mandibular; and (4) pleuro-coxal. These fell into 12 groups, based upon shape, density and pattern of tergal and femoral pegs, number of terga bearing pegs and presence of mandibular tusks bearing stridulatory tubercles. The plesiomorphic condition, consisting of spinose peg patches on 3 or more abdominal tergites rubbed by bands of pegs on the femur, is found in Hemiandrus spp. Apomorphically-derived tergal files have arisen separately in 3 other genera. In Hemideina, (2 species groups) well-formed files are very similar among species. In Deinacrida, (4 species groups), the trend is toward file reduction into one or 2 massive tergal ridges, and embellished femoral pegs. In the tusked wetas (2 gen., 2 spp.), one species has a crude file of many broken ridges. Stridulatory structures on mandibular tusks of both New Zealand species are unique, although the tusks appear to have their origin in African stenopelmatids. The plesiomorphic condition appears to have been deployed originally for defense stridulation (inter-specific communication). Additional intea-specific stridulatory communication developed in Hemideina and Deinacrida, using the apomorphic file and peg mechanisms. Here, the stridulation is associated with defense, calling, mating and disturbance behaviors.     

Ultrastructural cytochemistry of the sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae: Phlebotominae)

The sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae) were analyzed by cytochemical techniques. In adult males, the epithelium at the fourth abdominal tergite is modified into a glandular epithelium, with large columnar gland cells located side by side. The gland cell cytoplasm contains a large number of mitochondria and peroxisomes, the latter with positive (electron-dense) reaction for catalase, a typical peroxisomal enzyme marker. The gland cell cytoplasm also contains a central vacuolated area, with a large number of electron-lucent vacuoles, not limited by a unit membrane. In well-preserved preparations such vacuoles present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide (to detect lipids) resulted in positive reaction in these vacuoles, as well as in between the microvilli of the gland cells. Use of the osmium–potassium iodide (Os–KI) technique allowed to demonstrate the presence of several endoplasmic reticulum (ER) profiles, as expected in secretory cells. Our data suggest that ER, lipid droplets and peroxisomes are involved in the sand fly pheromone biosynthesis.     

cDNA cloning of an adult male putative lipocalin specific to tergal gland aphrodisiac secretion in an insect (Leucophaea maderae).

Lma-P22 is a cuticular surface protein specific to the tergal gland secretion of Leucophaea maderae adult males which is ingested by females just before copulation. The complete Lma-P22 cDNA sequence was determined by RT-PCR using primers based on Edman degradation fragments. The recombinant protein expressed in Escherichia coli was recognized by an anti-Lma-P22 antibody. Northern blot analysis indicates that the corresponding mRNA is transcribed only in the epidermis of male tergites. Sequence analysis indicated that Lma-P22 deduced protein belongs to the lipocalin family. Lipocalins are extracellular proteins which carry hydrophobic compounds and some of them can bind sexual pheromone in vertebrates. Lma-P22 is the first example of a lipocalin-like protein involved in insect sexual behavior.     

Sand fly specificity of saliva-mediated protective immunity in Leishmania amazonensis-BALB/c mouse model

Immune response of BALB/c mice to the salivary antigens of sand flies was found to vary with different species used, i.e. Phlebotomus papatasi, Phlebotomus sergenti and Lutzomyia longipalpis. Exposure of mice to bites of these sand flies elicits production of antibodies, which are largely specific to different saliva antigens previously identified as unique to the respective fly species. When immunized intradermally (i.d.) with salivary gland lysates (SGL) of L. longipalpis, BALB/c mice developed partial protective immunity against challenges in the contralateral ears with Leishmania amazonensis plus the gland lysates. Preimmunization of these mice with the lysates from the other two species was ineffective, further indicative of the specificity of saliva-mediated immune response. The partial protective immunity observed is significant, although it is not as dramatic as reported previously in a different sand fly-mouse model. There is a correlation of this immunity with a lower number of mononuclear and polymorphonuclear phagocytes at the site of parasite inoculation. Vector species-specificity of this immunity implies its elicitation by unique saliva antigen-an issue which requires attention when designing saliva-based vaccines against leishmaniasis.     

The salivary 5'-nucleotidase/phosphodiesterase of the hematophagus sand fly, Lutzomyia longipalpis [corrected

Salivary gland homogenates from adult female Lutzomyia longipalpis sand flies contain large amounts of 5'-nucleotidase and phosphodiesterase activities. Phosphodiesterase activity was found to be associated with 5'-nucleotidase in several independent experiments: (i) it coelutes with 5'-nucleotidase on a molecular sieving column, (ii) it coelutes with 5'-nucleotidase on a chromatofocusing column, and (iii) it has the same thermal inactivation kinetics as the 5'-nucleotidase activity. Additionally, both activities are independent of divalent cations, and both are decreased following a blood meal, suggesting that they reside in the same molecule. The role of salivary nucleotidases and purine nucleotides in blood-feeding by sand flies is discussed.     

Studies on populations of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in Brazil

Studies were performed on five Brazilian populations of Lutzomyia longipalpis: Salvaterra (PA), S?o José do Ribamar (MA), Canindé (CE), Natal (RN) and Gruta da Lapinha, Lagoa Santa (MG). No morphological differences were observed that could distinguish between these populations. Homogeneity tests showed that the allopatric populations display a certain heterogeneity and that the sympatric populations, with different patterns of spots, are homogeneous. The Student-Newman-Keuls test, represented by Euler-Venn diagrams, showed a disjunction between the populations from the north/northeast and the one from Gruta da Lapinha. Genetic distances between the four populations (excluding the Canindé population) were within the range of intrapopulational differences. The Gruta da Lapinha population displayed a heterozygotic deficiency that could be a consequence of high levels of inbreeding due to cryptic habits of living in a small cave. These results do not favor the hypothesis of a L. longipalpis species complex in Brazil, and the species should be considered high polymorphic.     

Ultrastructure of posterior sternal glands of Macrotermes annandalei (Silvestri): new members of the sexual glandular set found in termites (Insecta)

In female alates of Macrotermes annandalei, two types of abdominal glands are involved in the secretion of sex pheromone. Tergal glands are found at the anterior margin of tergites 6-10 and posterior sternal glands (PSGs) are located at the anterior margin of sternites 6-7. The cytological features of both types of glands are quite similar. The fine structural organization of PSGs is studied more precisely and described for the first time. The glandular cuticle is pitted with narrow apertures corresponding to the openings of numerous subcuticular pouches. Several Class 3 glandular units open in each pouch. One canal cell and one secretory cell make an individual glandular unit. The canal cell is enlarged apically and is connected with the other canal cells to form a common pouch. Based on the structural features found in these glands, we propose a common secretory process for PSGs and tergal glands. During the physiological maturation of alates inside the nest, secretory vesicles amass in the cytoplasm of secretory cells, while large intercellular spaces collapse the cuticular pouches. At the time of dispersal flight, pouches are filled with the content of secretory vesicles while intercellular spaces are sharply reduced. After calling behavior, no secretion remains in the glands and pouches collapse again, while secretory cells are drastically reduced in size. The structure and the secretory processes of PSGs and tergal glands are compared to those of abdominal sexual glands known in termites.     

Chemistry and morphology of the tergal gland of freeliving adult Aleocharinae (Coleoptera: Staphylinidae) and its phylogenetic significance

Abstract. Morphology and chemistry of the tergal gland, an abdominal defensive gland of the Staphylinidae subfamily Aleocharinae, is studied comparatively in twenty-two species of ten Central European tribes. To determine trends in the morphological evolution of the tergal gland the results are compared with well-known evolutionary trends of gland systems of other insects. The possible chemical evolution of the gland secretion is proposed by considering the biosynthesis of the different secretion compounds and by testing their biological efficiency on Calliphora larvae (irritancy, mortality). The morphological evolution probably proceeded from a small reservoir with only a few glandular cells D₁ (primitive) to a large and bilobed reservoir with a great number of glandular cells D₁ (advanced). In the chemistry of the quinone-containing gland secretion, long-chain fatty acids as solvents in primitive species were probably replaced by short-chain fatty acids (derived condition). Most advanced species also use hydrocarbons and aldehydes as solvents. With reference to these results, both a statistical (Jaccard-coefficient) and a cladistic analysis are performed to test previous ideas on the phylogeny of the Aleocharinae.     

Examination of the interior of sand fly (Diptera: Psychodidae) abdomen reveals novel cuticular structures involved in pheromone release: Discovering the manifold

The males of many species of New World Phlebotomines produce volatile terpenoid chemicals, shown in Lutzomyia longipalpis s.l. to be sex/aggregation pheromones. Pheromone is produced by secretory cells which surround a cuticular reservoir which collects the pheromone and passes it through a cuticular duct to the surface of the insect. The pheromone then passes through specialised cuticular structures on the abdominal surface prior to evaporation. The shape and distribution of the specialised structures are highly diverse and differ according to species. In this study we used SEM to examine the interior cuticular pheromone collection and transport structures of 3 members of the Lu. longipalpis s.l. species complex and Migonemyia migonei. We found a new structure which we have called the manifold which appears to be a substantial extension of the interior tergal cuticle connected in-line with the cuticular duct and reservoir. The manifold of the Campo Grande member of the complex is longer and wider than the Jacobina member whereas the manifold of the Sobral member was shorter than both other members of the complex. Overall, the secretory apparatus of the Sobral member was smaller than the other two. The manifold of M. migonei was very different to those found in Lu. longipalpis s.l. and was positioned in a pit-like structure within the tergal cuticle. The secretory reservoir was connected by a short duct to the manifold. Differences in the size and shape of the manifold may be related to the chemical structure of the pheromone and may have taxonomic value. Examination of the interior cuticle by SEM may help to locate the secretory apparatus of vector species where pheromonal activity has been inferred from behavioural studies but the external secretory structures or pheromones have not yet been found.     

External morphology of sensory structures of fourth instar larvae of neotropical species of phlebotomine sand flies (Diptera: Psychodidae) under scanning electron microscopy.

In the present study, some morphological structures of antennae, maxillary palps and caudal setae of fourth instar larvae of laboratory-reared phlebotomine sand flies (Lutzomyia longipalpis, L. migonei, L. evandroi, L. lenti, L. sericea, L. whitmani and L. intermedia) of the State of Ceará, Brazil, were examined under scanning electron microscopy. The antennal structures exhibited considerable variation in the morphology and position. A prominent digitiform distal segment has been observed only on the antenna of species of the subgenus Nyssomyia. The taxonomic relevance of this and other antennal structure is discussed. The papiliform structures found in the maxillae and the porous structures of the caudal setae of all species examined may have chemosensory function. Further studies with transmission electron microscopy are needed to better understand the physiological function of these external structures.     

Behavior of Somatic Cells Homozygous for Zygotic Lethals in DROSOPHILA MELANOGASTER

The behavior in genetic mosaics of 86 EMS-induced sex-linked lethals has been studied. Seventy-five percent of them are autonomous in gynandromorphs. Forty-three lethals nonviable in sex mosaics have been analyzed in X-ray-induced spots in the abdominal tergites and the imaginal wing derivatives. Of the lethals, 90.7% are homozygous viable in mosaic spots, and only 9.3% have been classified as epidermal cell lethal. Thus, the fraction of the Drosophila genome essential for cell viability has been estimated to be about 420 genes. The phenotypes at the cellular level of some cell-viable mutations altering cell parameters (mitotic orientation, differentiation, etc.) are described.     

Unusual location and structure of female pheromone glands in Theresimima (= Ino) ampelophaga Bayle-Berelle (Lepidoptera : Zygaenidae)

Morphological and behavioural studies strongly suggest that the sex pheromone glands of female Theresimima (= Ino) ampelophaga (Lepidoptera : Zygaenidae) are situated on the anterior part of the 3rd–5th abdominal tergites. The glandular epithelium consists of 2 cell types: gland cells and wrapping cells. The gland cells have a central microvilli-lined cavity which is in contact with the lumen of the hair. These hairs are exposed during the calling behaviour, and the pheromone is probably given off via pores in the scale wall.     

Age at death of the Neanderthal child from Devil's Tower, Gibraltar and the implications for studies of general growth and development in Neanderthals

This study combines traditional methods of assessing dental developmental status based upon modern human standards with new techniques based upon histological observations in order to reassess the age at death of the Gibraltar child from Devil's Tower. The results indicate that the most likely age of this individual at death was 3 years of age. This result is in agreement with an independent assessment of the age of the temporal bone of this specimen (Tillier, AM [1982] Z. Morphol. Anthropol. 73:125-148) and is concordant with dental developmental ages given for modern humans. Moreover, the fact that this specimen appears at the low end of the age scale for calcification stages in modern humans is also supportive of the findings of Legoux (Legoux, P [1970] Arch. Inst. Paleontol. Hum. Mem. 33:53-87) and Wolpoff (Wolpoff, MH [1979] Am. J. Phys. Anthropol. 50:67-114) that dental eruption schedules in Neanderthals were also accelerated. If the cranial bones from Devil's Tower are associated with the dental material, as we believe, they indicate a remarkably precocious brain growth in this individual, which is consistent with what is known about general growth and development in Neanderthals.