Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue.

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes' large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae.

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle - the Drosophila pupa - offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Food poisoning associated with ingestion of wild wasp broods in the upstream region of the Lancang river valley, Yunnan province, China.

INTRODUCTION: Food poisoning due to wild wasp broods ingestion has long been noted in the upstream region of the Lancang river valley, Yunnan province, China. This study describes the epidemiological and clinical features of the poisoning and possible causes. METHODS: Surveillance data collected between 2008 and 2016 were analyzed to produce demographic data on patients, information on clinical presentations, wasp species identification, and estimations of possible risk factors for symptomatic cases. RESULTS: Eleven poisoning events were associated with the ingestion of wild wasp broods, including 46 exposed persons with 31 symptomatic living cases and 8 deceased cases that were reported in the Yunnan province between 2008 and 2016. Poisoning cases were only detected in the upstream region of the Lancang river valley in the autumn. The severity of the symptoms was correlated with an evident dose-effect relationship regarding the quantity ingested. The mean latent period from wild wasp broods ingestion to the onset of the symptoms was 10 h for symptomatic living cases and 7 h for deceased cases, respectively. Both gastrointestinal and neurological symptoms were commonly observed in the poisoning cases. CONCLUSION: The toxin source may be indirectly caused by the wasp broods due to the prevalence of local poisonous plants, such as Tripterygium wilfordii Hook F, Tripterygium hypoglaucum Hutch and Vaccinium bracteatum Thunb. Educational programs at the start of wasp harvest season in September in the high-risk area should be carried out to reduce the incidence of poisonings.

Modeling the cost-effectiveness of insect rearing on artificial diets: A test with a tephritid fly used in the sterile insect technique.

We modeled the cost-effectiveness of rearing Anastrepha ludens, a major fruit fly pest currently mass reared for sterilization and release in pest control programs implementing the sterile insect technique (SIT). An optimization model was generated by combining response surface models of artificial diet cost savings with models of A. ludens pupation, pupal weight, larval development time and adult emergence as a function of mixtures of yeast, a costly ingredient, with corn flour and corncob fractions in the diet. Our model revealed several yeast-reduced mixtures that could be used to prepare diets that were considerably cheaper than a standard diet used for mass rearing. Models predicted a similar production of insects (pupation and adult emergence), with statistically similar pupal weights and larval development times between yeast-reduced diets and the standard mass rearing diet formulation. Annual savings from using the modified diets could be up to 5.9% of the annual cost of yeast, corn flour and corncob fractions used in the standard diet, representing a potential saving of US $27.45 per ton of diet (US $47,496 in the case of the mean annual production of 1,730.29 tons of artificial diet in the Moscafrut mass rearing facility at Metapa, Chiapas, Mexico). Implementation of the yeast-reduced diet on an experimental scale at mass rearing facilities is still required to confirm the suitability of new mixtures of artificial diet for rearing A. ludens for use in SIT. This should include the examination of critical quality control parameters of flies such as adult flight ability, starvation resistance and male sexual competitiveness across various generations. The method used here could be useful for improving the cost-effectiveness of invertebrate or vertebrate mass rearing diets worldwide.

Competition between the filth fly parasitoids Muscidifurax raptor and M. raptorellus (Hymenoptera: Pteromalidae).

Competition bioassays were conducted with the filth fly pupal parasitoids Muscidurax raptor (Girault & Sanders) and M. raptorellus (Kogan & Legner) (Hymenoptera: Pteromalidae) using house fly Musca domestica L. (Diptera: Muscidae) hosts at different host densities. Muscidifurax raptor had a significant impact on M. raptorellus when hosts were limiting in sequential parasitism tests. Fewer than six M. raptorellus adult progeny emerged from groups of 50 fly pupae that were parasitized by M. raptor at the same time or when M. raptor parasitism preceded M. raptorellus by 48 h, respectively, compared with 42-55 M. raptorellus progeny produced when this species was tested alone. Production of M. raptor was significantly lower when parasitism by this species was preceded by M. raptorellus (25) than when M. raptor was tested alone (43). When the two species parasitized hosts at the same time in different proportions at low host:parasitoid densities (5:1), M. raptorellus produced 13 progeny per parent female when it was the sole species present and fewer than two when M. raptor was present. No negative impact of M. raptorellus on M. raptor was observed. Neither species had a substantial effect on the success of the other at higher host:parasitoid densities.