Kairomones from Euschistus heros egg masses and their potential use for Telenomus podisi parasitism improvement.

Telenomus podisi Ashmead (Hymenoptera: Scelionidae) is the most important egg parasitoid of Euschistus heros (Fabricius) (Hemiptera: Pentatomidae), and its successful parasitism is related to their searching ability to find suitable hosts under a complex chemical environment using host-reliable cues. Thus, the objective of this study was to elucidate chemical substances on the external layer of E. heros eggs and report its potential kairomonal activity on T. podisi. We tested female wasps in olfactometer system to synthetic compounds obtained from a chemical identification of E. heros egg masses. The synthetic blend was also evaluated in parasitism tests under laboratory and semi-field conditions. We identified 31 substances from egg surface extracts, including monoterpenes, aldehydes and alkanes. Among those compounds, a synthetic solution including camphene, ß-pinene, limonene and benzaldehyde-induced chemotaxic behaviour on the wasps and increased the parasitism on E. heros eggs, either in laboratory or semi-field test, suggesting its potential use to T. podisi manipulation and parasitism improvement.

Male-Produced Sex Pheromone of Tibraca limbativentris Revisited: Absolute Configurations of Zingiberenol Stereoisomers and their Influence on Chemotaxis Behavior of Conspecific Females.

The rice stalk stink bug, Tibraca limbativentris, is an important rice pest in Brazil with a high invasive potential for Mexico and the USA. The sex pheromone of this species was previously identified as a combination of two stereoisomers of 1,10-bisaboladien-3-ol (zingiberenol), but the absolute configurations of these sesquiterpenes were not determined, neither were their effect(s) on T. limbativentris behavior evaluated. In this study, using two chiral columns, we compared retention times of the two natural 1,10-bisaboladien-3-ol stereoisomers from air-entrainment samples of male T. limbativentris with those of synthetic stereoisomers of 1,10-bisaboladien-3-ol. The results showed that T. limbativentris males produce (3S,6S,7R)-1,10-bisaboladien-3-ol (1) and (3R,6S,7R)-1,10-bisaboladien-3-ol (5) as their sex pheromone. Two new minor, male-specific components were also identified as cis and trans isomers of 2,10-bisaboladien-1-ol (sesquipiperitol). Y-tube olfactometer bioassays showed that the major (3S,6S,7R) isomer 1 was essential for attraction of T. limbativentris females, but the minor (3R,6S,7R) isomer 2 was not, nor did it show synergistic/antagonistic effects when added to the major isomer. The (1S,6S,7R) and (1R,6S,7R) stereoisomers of sesquipepiritol also attracted T. limbativentris females.

Conditioning Native Telenomus and Trissolcus (Hymenoptera: Scelionidae) Egg Parasitoids to Recognize the Exotic Brown Marmorated Stink Bug (Heteroptera: Pentatomidae: Halyomorpha halys).

Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug (BMSB), is a polyphagous fruit and vegetable pest from eastern Asia and now invasive in the United States and Europe. Earlier research revealed that the main volatile from hexane egg extracts of a western North American pentatomid, Euschistus conspersus Uhler, to which two native scelionid egg parasitoids, Telenomus podisi Ashmead and Trissolcus erugatus Johnson are highly attracted, was methyl (2E,4Z)-2,4-decadienoate, the major component of the conspecific male-produced aggregation pheromone. Conversely, extracts of BMSB eggs lacked the male-produced sesquiterpenoid H. halys pheromone components but contained C16,18,20 aldehydes (hexadecanal, octadecanal, and eicosanal); both egg-surface extracts of BMSB eggs and the C16,18,20 synthetic aldehyde blend repelled the female parasitoids. The goal of the present research was to manipulate egg-surface volatiles to induce the scelionid egg parasitoids to adopt BMSB eggs as a host. Here it was demonstrated that individual females of both parasitoids could be conditioned to H. halys egg-surface aldehydes in Y-tube olfactometer experiments, and this positive memory lasted 3-4 d. Furthermore, conditioned wasps successfully parasitized fresh H. halys eggs in no choice tests, and their offspring continued to successfully parasitize fresh H. halys eggs for 2-3 more generations. Eventually, both the individual and generational memories faded. Unconditioned female parasitoids were not attracted to H. halys egg extract or the synthetic blend of egg-surface aldehydes, nor did they parasitize BMSB eggs. Theoretical and practical implications of the research are discussed in the contexts of Hopkins' host selection principle, biological control, and instinct evolution.

Responses of Spodoptera frugiperda and Trichogramma pretiosum to Rice Plants Exposed to Herbivory and Phytohormones.

Damage caused by herbivorous insects and application of phytohormones can activate signaling pathways, which result in greater production of secondary metabolites, increasing plant defenses. This study aimed to evaluate the induced direct resistance (local and systemic) of rice plants caused by herbivorous insects and exogenous application of methyl jasmonate (MJ) and salicylic acid (SA) in the development of fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae). Moreover, indirect defenses through chemotactic responses of Trichogramma pretiosum (Riley) (Hymenoptera: Trichogrammatidae) submitted to the same treatments were investigated. Direct defense was evaluated by measuring weight gain of fall armyworm larvae fed with leaves of plants previously exposed to herbivores or treated with MJ (2 and 5 mM), SA (8 and 16 mM), or control. Indirect defense was verified by chemotactic behavior of wasps in two-choice olfactometer tests to plants exposed to herbivores and evaluated after 24, 48, and 72 h in comparison with undamaged ones, as well as plants treated with the same phytohormones contrasted with the control. The gain of weight was reduced in immature developmental stage of S. frugiperda fed in leaves previously damaged by fall armyworm and in newly formed leaves after damage to the plants, comparing with control. Leaves treated with MJ (2 mM and 5 mM) and SA (8 mM) were less eaten than those not treated. Parasitoids triggered a positive chemotactic behavior in rice plants that had been sprayed with same concentrations. This study showed that rice plants can activate direct and indirect defenses through an exogenous application of phytohormones.

Is It Possible to Manipulate Scelionidae Wasps' Preference to a Target Host?

Parasitoid host selection is mainly mediated by chemical cues, which can be adjusted by experience, changing their innate behavior. Therefore, this study evaluated if immature experience (pre-imaginal conditioning) on eggs and volatiles from different host eggs has influence on parasitism and chemotaxic behavior of Telenomus podisi Ashmead and/or Trissolcus basalis Wollaston (Hymenoptera: Scelionidae). Both wasp species were submitted to a multiple-choice parasitism test among Euschistus heros (Fabricius), Piezodorus guildinii (Westwood), and Nezara viridula L. (Hemiptera: Pentatomidae) (Hemiptera: Pentatomidae) egg masses. Eggs from these three stink bugs were equally offered to female parasitoids. After that, adults which emerged from each host were also exposed to parasitism in a multiple-choice test for up to an additional generation. Moreover, in olfactometer "Y," the behavior of innate and experienced T. podisi females to volatiles from hosts' egg extracts was tested, to study their learning and memory ability. The original host had influence on T. podisi parasitism; however, T. basalis always parasitized more N. viridula eggs independently of its last rearing host. Innate T. podisi females responded positively to E. heros and P. guildinii egg volatiles, but this behavior was not observed in N. viridula. When T. podisi females were experienced on egg volatiles from a new host, they showed significant learning and memory ability for the specific host volatile for, at least, 24 h. Experienced wasps responded positively to N. viridula and through this result we have evidences about the possibility to manipulate wasp's preferences to a specific target host.

Chemical Composition of Alphitobius diaperinus (Coleoptera: Tenebrionidae) Abdominal Glands and the Influence of 1,4-benzoquinones on its Behavior.

This study aimed to identify and quantify the compounds present in the abdominal glands of Alphitobius diaperinus Panzer, 1797 (Coleoptera: Tenebrionidae) and to evaluate the influence of these compounds on its behavior. The extraction of volatiles present in the abdominal glands was made by dissection (10 individuals per sex) and by air entrainment (200 insects per sex), and they were analyzed by gas chromatography-flame ionization detector, gas chromatograph-mass spectrometer, and gas chromatograph-electroantennography detector (GC-EAD). The influence of these volatiles on the behavior of conspecifics was evaluated in a four-arm olfactometer. Twenty-three compounds were identified from male and female abdominal gland extracts, of which six were quinones: the 2-methyl-1,4-benzoquinone and the 2-ethyl-1,4-benzoquinone were the major components, and 1,4 benzoquinone and three hydroquinones were registered for the first time for this species. The GC-EAD analysis using the crude extracts from abdominal glands showed that male and female antennae responded to the three major benzoquinones. For the olfactometer bioassays, both genders were repelled either by the abdominal gland extracts or by synthetic solutions containing the three benzoquinones. The results suggest that the 1,4-benzoquinones play a role as a repellent to A. diaperinus.

Influence of original host on chemotaxic behaviour and parasitism in Telenomus podisi Ashmead (Hymenoptera: Platygastridae).

The egg parasitoid Telenomus podisi is a natural control agent of pentatomids, including Euschistus heros and Tibraca limbativentris, and success of parasitism is dependent upon the parasitoid finding the host. We tested the influence of host egg volatiles and the synthetic sex pheromone (zingiberenol) of T. limbativentris on chemotaxic behaviour of T. podisi, as well as, the impact of the original host on parasitoid selection. We used mated female T. podisi (48 h old) that emerged from the eggs of T. limbativentris or E. heros. The bioassays related to chemotaxy were performed in a Y-tube olfactometer and, to parasitism success, in laboratory and semi-field conditions. Telenomus podisi females that emerged from either the stink bug eggs, chose the pheromone more than control, or the pheromone plus eggs of E. heros in the semi-field bioassay, led to greater parasitism. Females that emerged from E. heros eggs chose egg volatiles from their original host rather than those from T. limbativentris, while females emerging from T. limbativentris, chose the egg volatiles of both hosts equally. When T. limbativentris was the original host, T. podisi females parasitized T. limbativentris over E. heros, while those emerging from E. heros exclusively parasitized E. heros eggs. These results demonstrated that T. podisi is more likely to parasitize the host in which it developed and that the original host can exert influence on the choice by those parasitoids. Understanding how the factors that mediate host-parasitoid communication are interrelated can help biological control programmes establish more effective and reliable tools with T. podisi.

Herbivory-induced plant volatiles from Oryza sativa and their influence on chemotaxis behaviour of Tibraca limbativentris Stal. (Hemiptera: Pentatomidae) and egg parasitoids.

The rice stem bug, Tibraca limbativentris Stal. (Hemiptera: Pentatomidae) is one of the most important pests of rice crops, especially irrigated crops. Plant defence strategies against these bugs may involve the emission of chemical compounds, which are released following herbivore attacks, directly or indirectly harming pest performance. The aim of this study was to evaluate the influence of constitutive and herbivory-induced volatiles from rice plants (Oryza sativa L.) on the behavioural responses of T. limbativentris adults and egg parasitoids Trissolcus basalis (Wollaston) and Telenomus podisi (Ashmead) (Hymenoptera, Platygastridae). Plant volatiles were collected from undamaged plants of the rice cultivar IRGA 424 and from plants that suffered herbivory by five males or five females of T. limbativentris. Air-entrainment extracts were analysed by GC-flame ionization detector and GC-MS, and insect responses evaluated in a 'Y' olfactometer. T. limbativentris feeding damaged on rice plants induced the release of 16 volatiles compounds in a higher amounts compared to undamaged plants The main compounds induced were (E)-2-hexenal, (E)-2-octen-1-ol, methyl salicylate and α-muurolene. Female bugs were significantly attracted to air-entrainment extracts containing volatiles from undamaged plants compared with air-entrainment extracts containing volatiles emitted from plants damaged by T. limbativentris, whereas males showed no preference. Telenomus podisi females were significantly attracted to volatiles from air-entrainment extracts of plants damaged by females, whereas T. basalis showed no preference. These results suggest that rice plants may be emitting defence compounds, which could be avoided by T. limbativentris females and also acted indirectly by attracting natural enemies.

Effect of dose and exposure to diatomaceous earth on different insects in stored corn / Efeito da dose e exposição à terra de diatomácea de diferentes insetos em milho armazenado

Sitophilus zeamais and Tribolium castaneum are important insect pests of stored grain and their damage corresponds to approximately 25% of post-harvest losses in Brazil. The methods used for the control of these pests notably include the use of post-harvest inert dusts. Therefore, the objective was to assess the mortality of adult S. zeamais and T. castaneum with the use of diatomaceous earth (DE) at different dosages and durations. The treatments with diatomaceous earth were 1,000 and 2,000 GT-1 and control (no application). Insects (10 each species) were placed in 300-ml plastic containers containing 100 g of corn kernels with different moisture contents (12, 14 and 16% wb). Infestations occurred 1 hour, 10 and 20 days after the application of TD. Mortality was evaluated at 30 and 60 days. There were five replicates per treatment. There was a higher mortality for the species S. zeamais than for T. castaneum, and this morality rate increased in direct relation to insect exposure time. It was concluded that T. castaneum has greater tolerance and that the more time the insects are in contact with treated grain the greater the mortality.

Sitophilus zeamais e Tribolium castaneum são importantes insetos-pragas de grãos armazenados e seus danos correspondem a aproximadamente 25% das perdas na pós-colheita do Brasil. Entre as formas de controle destaca-se o uso de pós-inertes. Sendo assim, o objetivo deste trabalho foi avaliar a mortalidade de adultos de S. zeamais e T. castaneum com uso de terra de diatomácea (TD) em diferentes dosagens e períodos de exposição. Os tratamentos com terra de diatomácea foram: 1.000 e 2.000 g.t-1 e controle (sem aplicação). Os insetos (10 de cada espécie) foram acondicionados em recipientes plásticos de 300 mL, contendo 100 g de grãos de milho com diferentes umidades (12, 14 e 16% b.u.). As infestações ocorreram 1 hora, 10 e 20 dias após a aplicação da TD. Avaliou-se a mortalidade aos 30 e 60 dias. Foram realizadas cinco repetições/tratamento. Obteve-se maior mortalidade para a espécie S. zeamais do que T. castaneum, sendo que ela aumenta conforme o tempo de exposição dos insetos. Conclui-se que a espécie T. castaneum apresenta maior tolerância e que quanto maior tempo os insetos estiverem em contato com grãos tratados maior será a mortalidade.

Diapause initiation and alterations in the life cycle of Grapholita molesta (Busck) (Lepidoptera: Tortricidae) as induced by photoperiodic conditions.

Grapholita molesta (Busck) is one of the major pests of Rosaceae, causing significant damage to buds and fruits. In Southern Brazil, its population density is reduced during Rosaceae dormancy months. The present study evaluated the influence of different photoperiods (L:D) (10:14, 11:13, 12:12, 13:11, 14:10 and 16:8) at 25 ± 1ºC and 60 ± 10% RH on diapause induction of G. molesta eggs, larvae, prepupae, and pupae. The effects of the photoperiod on the life cycle of non-diapausing insects and on the second generation were also assessed. Prepupal diapause was observed only when eggs and neonates (≤ 12h-old larvae) were exposed to photophases from 10h to 14h long. Development of non-diapausing individuals and those from the second generation tended to be longer in photophases between 10h and 14h long.

Diapause initiation and alterations in the life cycle of Grapholita molesta (Busck) (Lepidoptera: Tortricidae) as induced by photoperiodic conditions

Grapholita molesta (Busck) is one of the major pests of Rosaceae, causing significant damage to buds and fruits. In Southern Brazil, its population density is reduced during Rosaceae dormancy months. The present study evaluated the influence of different photoperiods (L:D) (10:14, 11:13, 12:12, 13:11, 14:10 and 16:8) at 25 ± 1ºC and 60 ± 10 percent RH on diapause induction of G. molesta eggs, larvae, prepupae, and pupae. The effects of the photoperiod on the life cycle of non-diapausing insects and on the second generation were also assessed. Prepupal diapause was observed only when eggs and neonates (< 12h-old larvae) were exposed to photophases from 10h to 14h long. Development of non-diapausing individuals and those from the second generation tended to be longer in photophases between 10h and 14h long.

Morphometry and distribution of sensilla on the antennae of Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae).

Antennal sensilla of Anastrepha fraterculus (Wied.) were examined using scanning electron microscopy. In the flagellum, there are trichoid, basiconic, clavate type I and II, and styloconic sensilla and microtrichia. Only microtrichiae and chaetica sensilla were observed in the scape and pedicel. The number of sensilla in the flagellum was similar between sexes. At the apex there was a higher density of trichoid and an absence of clavate sensilla, while basiconic sensilla were more abundant in the proximal region.

Morphometry and distribution of sensilla on the antennae of Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae)

Antennal sensilla of Anastrepha fraterculus (Wied.) were examined using scanning electron microscopy. In the flagellum, there are trichoid, basiconic, clavate type I and II, and styloconic sensilla and microtrichia. Only microtrichiae and chaetica sensilla were observed in the scape and pedicel. The number of sensilla in the flagellum was similar between sexes. At the apex there was a higher density of trichoid and an absence of clavate sensilla, while basiconic sensilla were more abundant in the proximal region.

Discrimination of Spartocera dentiventris (Berg, 1884) (Hemiptera: Coreidae) eggs by Gryon gallardoi (Brèthes, 1913) (Hymenoptera: Scelionidae).

This paper aimed to evaluate the stimuli that orient Gryon gallardoi females to the eggs of Spartocera dentiventris and allow their discrimination. Using a four-arm olfactometer connected to four odor sources--S. dentiventris eggs; hexane washed eggs; tobacco leaves; and tobacco leaves with eggs--the arrestment of female parasitoids with previous oviposition experience and without was individually verified. The time of permanence in each odor field was registered for a period of 15 minutes. Host discrimination, regarding age and previous parasitism, was tested using arenas, where females were exposed for 30 minutes, individually, to egg groups with different treatments. To evaluate age discrimination, three groups of eggs, previously washed with hexane, were employed in the following conditions: one day-old eggs; one-day brushed with 12-days-old egg extract; 12-days-old eggs with extract of one day-old eggs; and control (washed eggs, one day-old). The same procedure was done using five and eight days-old eggs jointly with control. Age-dependent egg discrimination was verified exposing four egg groups in the following treatments: parasitized, parasitized and washed with hexane, not parasitized, and not parasitized with extract of parasitized eggs. Olfactometer tests showed that inexperienced females remained more time next to tobacco leaves when compared to experienced ones. Experienced females responded to odors that emanated from eggs. Egg extracts did not promote age discrimination; however, non parasitized eggs, with extract of parasitized eggs were partially avoided. The results obtained indicate that G. gallardoi females might modify their responses upon contact with host. This fact suggests learning occurs; however, the acceptance and discrimination of host may be influenced by a complex array of stimuli, difficult to evaluate in isolation.

Discrimination of Spartocera dentiventris (Berg, 1884) (Hemiptera: Coreidae) eggs by Gryon gallardoi (Brèthes, 1913) (Hymenoptera: Scelionidae)

This paper aimed to evaluate the stimuli that orient Gryon gallardoi females to the eggs of Spartocera dentiventris and allow their discrimination. Using a four-arm olfactometer connected to four odor sources - S. dentiventris eggs; hexane washed eggs; tobacco leaves; and tobacco leaves with eggs - the arrestment of female parasitoids with previous oviposition experience and without was individually verified. The time of permanence in each odor field was registered for a period of 15 minutes. Host discrimination, regarding age and previous parasitism, was tested using arenas, where females were exposed for 30 minutes, individually, to egg groups with different treatments. To evaluate age discrimination, three groups of eggs, previously washed with hexane, were employed in the following conditions: one day-old eggs; one-day brushed with 12-days-old egg extract; 12-days-old eggs with extract of one day-old eggs; and control (washed eggs, one day-old). The same procedure was done using five and eight days-old eggs jointly with control. Age-dependent egg discrimination was verified exposing four egg groups in the following treatments: parasitized, parasitized and washed with hexane, not parasitized, and not parasitized with extract of parasitized eggs. Olfactometer tests showed that inexperienced females remained more time next to tobacco leaves when compared to experienced ones. Experienced females responded to odors that emanated from eggs. Egg extracts did not promote age discrimination; however, non parasitized eggs, with extract of parasitized eggs were partially avoided. The results obtained indicate that G. gallardoi females might modify their responses upon contact with host. This fact suggests learning occurs; however, the acceptance and discrimination of host may be influenced by a complex array of stimuli, difficult to evaluate in isolation.

Este trabalho objetivou avaliar os estímulos que direcionam as fêmeas Gryon gallardoi aos ovos de Spartocera dentiventris e promovem discriminação deles. Através de um olfatômetro de quatro braços conectado a quatro fontes de odor (ovos de S. dentiventris não lavados, ovos lavados com hexano, folhas de fumo e folhas de fumo com ovos) verificou-se, individualmente, o arrestamento dos parasitóides, experientes em oviposição e não experientes, registrando-se o tempo de permanência em cada campo de odor por 15 minutos. Para avaliar a discriminação do hospedeiro, em relação à idade e parasitismo prévio, utilizaram-se arenas, onde as fêmeas foram expostas por 30 minutos, individualmente, a grupos de ovos com diferentes tratamentos. Para a avaliação da idade, utilizaram-se três grupos de ovos, lavados com hexano, nas seguintes condições: ovos de um dia de idade pincelados com extrato de ovos de 12 dias, ovos de 12 dias com extrato de ovos de um dia e controle (ovos lavados de um dia). O mesmo procedimento foi feito utilizando-se ovos de cinco e oito dias de idade, juntamente com o controle. A discriminação de ovos previamente parasitados foi verificada expondo quatro grupos de ovos com os seguintes tratamentos: parasitados, parasitados e lavados com hexano, não parasitados e não parasitados com extrato de ovos parasitados. Testes em olfatômetro demonstraram que as fêmeas inexperientes permaneceram mais tempo junto às folhas de fumo que as experientes. As fêmeas experientes responderam aos odores oriundos dos ovos. Os extratos de ovos não promoveram a discriminação da idade, porém ovos não parasitados, impregnados com extrato de parasitados, foram parcialmente evitados. Os resultados deste trabalho apontam que as fêmeas de G. gallardoi podem modificar suas respostas diante do contato prévio com o seu hospedeiro, sugerindo aprendizagem, porém a aceitação e a discriminação do hospedeiro podem ser influenciadas por uma complexa combinação de estímulos,...

Hepatic granulomas in canine visceral leishmaniasis and clinical status

The histopathological description of intralobular hepatic granulomas in animals with a defined clinical status (asymptomatic, oligosymptomatic and symptomatic animals) was reported. Seventy-one mongrel dogs naturally infected with Leishmania chagasi were obtained from two Brazilian endemic areas: João Pessoa, PB and Belo Horizonte, MG. The hepatic parasite load was determined and compared to granuloma formation. Liver fragments from all infected animals showed remarkable leishmaniotic granulomatous inflammatory reaction. Granulomas with variable size were constituted by macrophages (parasitized or not with amastigotes of L. chagasi), some epithelioid cells, small numbers of lymphocytes, plasma cells, and rare neutrophils. Asymptomatic dogs had higher numbers of granulomas than oligosymptomatic and symptomatic animals from both geographical regions. However, the average diametric size of granulomas was very heterogeneous in all groups, independently of the geographic region (P>0.05). Parasite tissue load did not show any difference among liver fragments of all animals, especially when considering the defined clinical status and/or their geographic origin

Descreve-se a formação de granulomas hepáticos na leishmaniose canina em animais com classificação clínica definida - assintomáticos, oligossintomáticos e sintomáticos. Setenta e um animais, sem raça definida e naturalmente infectados com Leishmania chagasi, foram obtidos de duas regiões endêmicas brasileiras: João Pessoa, PB e Belo Horizonte, MG. A carga parasitária tecidual foi determinada mediante emprego do Leishmania Donovani Units (LDU) e comparada com a formação de granulomas hepáticos. Fragmentos de fígado de todos os animais infectados mostraram reação granulomatosa notadamente leishmaniótica. Granulomas de variáveis tamanhos eram constituídos por macrófagos, parasitados ou não com formas amastigotas de L. chagasi, algumas células epitelióides, pequeno número de linfócitos e plasmócitos, e raros neutrófilos. Cães assintomáticos apresentaram maior número de granulomas do que os animais oligossintomáticos e sintomáticos, em ambas as regiões geográficas. As médias dos diâmetros foram heterogêneas em todos os grupos, independente da região geográfica (P>0,05). Quanto ao parasitismo (LDU), não houve diferença entre as amostras de fígado, especialmente quando se consideraram a classificação clínica e a região geográfica

Survival of tubercle bacilli in heat-fixed and stained sputum smears.

We used a slide culture technique to detect tubercle bacilli surviving in sputum smears (n=46) after conventional heat fixation and Ziehl-Neelsen staining. In all heat-fixed sputum smears, tubercle bacilli survived after time 0 (n=22), 24 h (n=7), 48 h (n=7), 72 h (n=4), and seven days (n=6). None of the stained sputum smears showed growth on slide cultures. Viable tubercle bacilli remaining in heat-fixed sputum smears for at least seven days may present an infection risk to laboratory staff. Thus, sputum smears should be stained immediately by the Ziehl-Neelsen method or stored in a safe container to avoid transmission of tuberculosis.

Comparison of the molecular, antigenic and ATPase determinants of fast myosin heavy chains in rat and human: a single-fibre study.

Combined methodologies of histochemistry, immunohistochemistry, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), reverse transcriptase polymerase chain reaction (RT-PCR) and a histochemical method specific for myofibrillar ATPase (mATPase) of the type IIX myosin heavy chain (MyHC) isoform were used to study human and rat single fibres to examine the homology between type II MyHC isoform-based fibres of both species. We demonstrate that human type II fibres exhibit antigenic mATPase and 3'-untranslated region (3'-UTR) sequence determinants homologous to the IIA and IIX but not the IIB MyHC isoforms of the rat. Both immunolabelling with anti-MyHC monoclonal antibodies and the mATPase method used with frozen sections confirmed that all human type II fibres express type IIA and/or type IIX MyHC. Quantitative immunohistochemistry failed to recognize human fibres with antigenic characteristics corresponding to hybrid IIXB MyHC-based fibres. Ca2+-stimulated maximum myosin ATPase activity, determined by quantitative histochemistry, revealed that human IIX fibres (with an optical density or OD = 0.707) display enzyme activity which is comparable to that of the rat type IIX (OD = 0.687) but lower than that of the rat type IIB fibres (OD = 0.836). The results do not support the notion that MyHC IIB is expressed in human limb muscles, even in hybrid fibres. We conclude that human type II fibres have been misclassified in numerous previous publications and that this has important implications in attempts to compare the physiological characteristics of fibre types, particularly when animal models are used.