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Rat-tailed larvae of the syrphid species Palpada scutellaris (Fabricius, 1805) are documented causing an enteric human myiasis in Costa Rica. This is the first time that the genus Palpada is recorded as a human myiasis agent. We report a 68-year-old woman with intestinal pain and bloody diarrhea with several live Palpada larvae present in the stool. Using molecular techniques (DNA barcodes) and both electronic and optical microscopy to study the external morphology, the preimaginal stages of the fly were unambiguously identified. An identification key to all syrphid genera actually known as agents of human and animal myiases is provided for larvae, puparia, and adults. Moreover, a critical world review of more than 100 references of Syrphidae as myiasis agents is also given, with emphasis on the species with rat-tailed larvae.

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Non-aquatic reproductive modes have evolved among frogs possibly favored by some advantages such as the avoidance of aquatic predators. These reproductive modes, however, make the egg clutches susceptible to terrestrial predators, among which Diptera larvae are some of the most harmful. The present work reports the predation by phorid flies of 22 egg clutches of Phyllomedusa iheringii Boulenger in the South of Brazil. Phorid specimens were identified as Megaselia bruchiana (Borgmeier & Schmitz) and Megaselia necrophaga (Enderlein), species that were reported previously to be associated with ants and dead beetles, respectively. Frog-feeding in these species is hypothesized to be use of an alternative seasonal food source. We amend the diagnoses of both Megaselia species and provide new illustrations to facilitate their identification. We also describe the male of M. bruchiana for the first time and associate males with females of both species.

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Sarconesiopsis magellanica (Le Guillou) (Diptera: Calliphoridae) is a necrophagous fly that is important in both human and veterinary medicines. This insect has been registered in Colombia as a biological indicator in estimating post-mortem interval. Insect cell cultures are an important biotechnological tool for basic and applied studies, and cell cultures derived from S. magellanica embryonic tissues are described in this study. S. magellanica embryonated eggs were taken for tissue explants. These were seeded in L-15, Grace/L-15, Eagle MEM, MM, VP12, MM/VP12, and Schneider culture media. The morphological, cytogenetic, biochemical, and molecular characteristics of the cell cultures were examined. Cell growth was achieved in the L15, Grace/L15, and Schneider culture media, and the confluent monolayers were obtained 8, 10, and 19 days after the embryonated eggs were explanted. However, the Schneider medium was the most efficient to develop the subcultures, and 21 passages have been maintained. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer and in the subcultures there was greater cell morphology uniformity, fibroblastoid types being predominant. Cultured cells had a chromosomal number of 12, and the karyotypic complement consisted of five pairs of somatic chromosomes and one sexual pair. The cell culture isozyme patterns of S. magellanica coincided with adult samples from the same species. The molecular analysis, using RAPD-PCR, demonstrated the authentication of the cell cultures of this fly and their differentiation from other cultures derived from two sand flies species. This cell line is a new in vitro model that will be used in biomedical and biotechnological studies.

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Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 µg/mL.

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The spermatozoa of Chrysomya megacephala are similar to those described for other Brachycera. In this species, the spermatozoa are long and thin, measuring about 590microm in length, of which the head region measures approximately 60microm. The head includes a monolayered acrosome with electron-lucid material, and the shape of the nucleus, in cross-sections, varies from circular to oval with completely condensed chromatin. The centriole was observed in the zone of flagellar implantation, below the "peg" region. In the region of overlap, the followings structures are observed: nucleus, centriolar adjunct, mitochondrial derivatives and axoneme. The two mitochondrial derivatives are of different lengths but similar diameter. The axoneme is of a conventional insectan type with a 9+9+2 microtubular arrangement, with accessory tubules flanked by the electron-dense intertubular material. The male internal reproductive tract consists of testis, vas deferens, seminal vesicle, accessory glands and ejaculatory duct.

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The midgut of adult female Anopheles aquasalis presents a narrow anterior or thoracic region and a distensible posterior or abdominal region constituted by the epithelium formed by a cell layer whose apical portion presents microvilli and the basal portion, a basal labyrinth. The thoracic region revealed heterogeneous cellular staining affinity mainly by the presence of acidic components. The ultrastructural aspect showed columnar cells with the presence of the vesicle, mitochondria, endoplasmic reticulum and secreting cells. The abdominal region of the midgut revealed an irregular epithelium whose cells presented a basophilic cytoplasm and acidophil granules. It was also found secreting and/or basal cells with narrow cytoplasm. The ultrastructural observation of this region demonstrated cells with evident nucleus, endoplasmic reticulum and mitochondria. Larger vesicles and small granules were found distributed throughout the cytoplasm. The basal lamina that supports the epithelium presented a generally irregular aspect and the muscle fibers have longitudinal and circular organization and were found separating the epithelium from the haemocel. This study will contribute to analyses on the vector mosquito-parasite interaction mechanism in this specimen.

La seccion media del intestino de la hembra de Anopheles aquasalis presenta una estrecha region anterior o toráxica y una region posterior o abdominal constituida por el epitelio formado por una camada de células cuya porcion apical presenta microvilosidades y la porcion basal presenta un laberinto basal. La region toráxica reveló afinidad de tintura celular principalmente para componentes acídicos. El aspecto ultra estructural mostró células columnares con la presencia de la vesícula, mitocondrias, retículo endoplasmático y células secretoras. La region abdominal del intestino medio reveló un epitelio irregular con células con citoplasma basófilo y granulos acidófilos. También se encontraron células secretoras y/o básales con citoplasma estrecho. La observacion ultra estructural de la region mostró células con núcleos, retículo endoplasmático y mitocondrias evidentes. Vesículas largas y granulos pequeños fueron encontrados distribuidos por todo el citoplasma. La lámina basal que apoya el epitelio presentó un aspecto irregular y las fibras musculares tienen organizacion longitudinal y circular y separan el epitelio del hemocele. Este estudio contribuirá al análisis del mecanismo de interaccion entre el mosquito y el parásito en este espécimen.

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Intrinsic bent DNA sites were identified in the 4289 bp segment encompassing the replication zone which directs DNA amplification and transcription of the C3-22 gene of Rhynchosciara americana. Restriction fragments showed reduced electrophoretic mobility in polyacrylamide gels. The 2D modeling of the 3D DNA path and the ENDS ratio values obtained from the dinucleotide wedge model of Trifonov revealed the presence of four major bent sites, positioned at nucleotides -6753, -5433, -5133 and -4757. Sequence analysis showed that these bends are composed of 2-6 bp dA.dT tracts in phase with the DNA helical repeat. The circular permutation analysis permitted the verification that the fragments containing the bending sites promote curvature in other sequence contexts. Computer analyses of the 4289 bp sequence revealed low helical stability (DeltaG values), negative roll angles indicating a narrow minor groove and a putative matrix attachment region. The data presented in this paper add to information about the structural features involved in this amplified segment.

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INTRODUCTION: Cell cultures from insects are a useful methodology in technological and biomedical studies. OBJECTIVE: The present work was aimed at obtaining and characterizing cell cultures derived from Aedes aegypti embryonic tissues. MATERIALS AND METHODS: Embryonated eggs were used for embryonic tissue explants in L-15/Grace and MMNP12 culture media, supplemented with 20% fetal bovine serum and a mixture of 1% antimycotic and antibiotics, at a pH ranging from 6.8 to 7.0. The incubation temperature was 28 degrees C; a CO2 atmosphere was not required. RESULTS: Cell growth was obtained in L-15/Grace medium three weeks after embryonic tissues explants. Six months were required for achieving a confluent monolayer. Twenty-eight serial cell subcultures were carried out from August 2003 to June 2004. Cell morphology was characterized as epithelial in the later subcultures. Karyotype morphometry as well as molecular and isozymatic profiles were established. The cultures were compared with adult samples from the species taken from the same colony and with cell lines derived from other insects. DISCUSSION AND CONCLUSIONS: These cells are an important in vitro system in applied and basic research.

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N-Beta-Alanyldopamine (NBAD) is the primary catechol tanning agent precursor in typical brown or yellow insect cuticle. The insect integument enzyme responsible for the synthesis of NBAD was reported to be expressed solely in the epidermis, and only at the time of cuticle sclerotization. However, in this study we demonstrate directly that the enzyme also is expressed in a constitutive manner in the neural system of insects. The requirements and kinetic parameters of the brain-associated enzyme appear similar to those of the epidermis-associated enzyme in Ceratitis capitata. The brain-associated enzyme also was able to catalyze the in vitro synthesis of N-beta-alanylnorepinephrine (NBANE) and beta-alanyl derivatives of other biogenic amines. A melanic mutant of C. capitata, niger, was unable to conjugate beta-alanine with dopamine or other amines in either the epidermis or the brain. This result strongly supports the idea that these enzymes actually are expressed from a single gene and that differences in regulation must exist that account for the constitutive expression in the neural system. Similar results were obtained in Drosophila melanogaster and other insects. From these data, a number of questions arise about the role of beta-alanyl derivatives of biogenic amines and other compounds in insect brain and similarly, in the mammalian CNS.

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In this work, we present biochemical and morphological evidence that the final steps of programmed cell death (PCD) in the salivary glands of the inferior Diptera, Bradysia hygida, present apoptotic characteristics. In B. hygida, elimination of salivary glands is preceded by the establishment of a typical pattern of protein synthesis; increase in caspase activity; decrease in cell volume; nuclear pyknosis; nuclear DNA breakage; changes in the actin cytoskeleton; and most importantly, destruction of giant cells via formation of apoptotic bodies containing broken DNA or cytoplasm remains. Thus, elimination of B. hygida salivary glands by this process suggests that such mode of PCD is also involved in the destruction of entire organs in insects and, therefore, adds more complexity to the regulation of tissue elimination during development.

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