Mz16a

Hand specimen photographs of Plesiosiro madeleyi are available in the redescription of Dunlop (1999) (link). No comparable modern photographs of Goniotarbus angulatus exist. Accordingly a plate of hand specimen photographs is published herein showing the holotype, and only known specimen (NHM In 22838: Fig. 2). This was studied and photographed using a Leica MZ16A stereomicroscope and incident light. Photographs taken at multiple focal depths were combined using the software CombineZM (see Bercovici, Hadley & Villanueva-Amadoz, 2009 ). Photographs of the whole fossil—which was too large for the field of view—were created by manually stitching sections using the open source raster graphics editor GIMP 2.8, and figures were assembled in Inkscape 0.48. For comparative purposes specimens of a related species (Petrunkevitch, 1949 ), Goniotarbus tuberculatus (NHM In 31249, NHM In 18340, and NHM In 22840), were also studied.

We used two high-end approaches that were already available in-house, a Leica MZ16A set-up and a Leica Z6APO. These solutions will be compared to the Canon-Cognisys set-up we describe above.

This study is based on material collected in 2010 by R.C. Drewes. Some additional samples were obtained from the Muséum national d’Histoire naturelle (MNHN), Paris, France and the Royal Museum for Central Africa (MRAC), Tervuren, Belgium.
All samples are stored in 70% ethanol. Photographs were made with a Leica DFC 500 digital camera mounted on a Leica MZ16A stereo microscope. Images were processed with a Leica Application Suite program. Specimens for scanning electron microscopy (SEM) were air-dried, mounted on aluminium stubs, coated with gold and studied using a JEOL JSM-6480LV scanning electron microscope.
The terminology used to describe the gonopod conformations follows that of Hoffman (2008).

The identification of the fiber and weaving pattern was performed with a light microscope (a low-power Leica MZ 16A and a high-power Leica DVM5000, respectively). A Y172 (Shanghai Precision Instruments Co., Ltd., Shanghai, China) Hastelloy slicer was used to observe the cross-section characteristics of the fiber. A morphology analysis was carried out by a high-vacuum Jeol JSM-6360LV scanning electron microscope (SEM) at an acceleration voltage of 8 kV and a working distance of 24 mm. The sample was gold-coated. UV imaging for the curtain was undertaken with a Canon camera at maximum exposure only under UV radiation in a darkroom.

Bees necropsied (usually 10 at a time) were removed from their storage bottles and allowed to dry slightly on a paper towel. We made two shallow incisions along the lateral sides of a bee’s abdomen, in the pleural region, using forceps and micro-scissors (EMS high-precision tweezers, style 5B with extra fine and bent tips; straight Dumont positive-action tweezers, Style SS; Vannas Capsulotomy scissors with 5 mm straight blade). Incisions started at the posterior end of the abdomen and ended near the petiole. Unless otherwise noted, once the incisions were made, we detached the abdomen from the thorax and placed it in a Petri-dish containing 70% ethanol to facilitate tissue separation. Under a stereo microscope (Leica MZ16 A), we removed abdominal sclerites, clearing away the sternites and tergites from around the gastro-intestinal tract until the internal tissues were completely visible. Following Snodgrass[35 ], we carefully examined each bee’s abdominal cavity, gastrointestinal tract, and sting region for gross lesions and other symptoms, and scored 17 different visible conditions as listed in Table 1.

Confocal images were generated as previously described (Arkhipova et al., 2012 (link)) with a Zeiss LSM Exciter5 microscope equipped with 25 mW Argon laser (468, 488 and 514 nm), 1 mW 543 nm He-Ne laser, and a 5 mW 633 nm He-Ne laser. Fluorescence detection was performed with the following filters: BP 505-550 (eGFP and Alexa 488), LP 560 (E2Crimson, mCherry) and LP 650 (Alexa 633, Click-IT 647). Additional confocal images were generated with a Leica TCS SP5 II Laser using 488 nm and 561 nm excitation wavelengths. Fluorescence detection was performed a bandwidth PMT 500 to 550 nm and a Hyd 3 bandwidth 600 to 650 nm. Adobe Photoshop CS5 was used for image arrangement and adjustment. Epifluorescence images were taken at the Leica MZ16A stereomicroscope using either a Leica DFC320 camera or a Zeiss AxioCam ICm1 camera.