Evo 40

The specimens were collected by bottom trawling from unexplored habitats down to 70 m deep in the middle mesophotic zone (from 40 to 150 m), on board of the Smithsonian Tropical Research Institute R/V Urracá along the north and central Pacific coast, from Santa Elena Bay to the Nicoya Gulf.
The specimens were fixed in 70% ethanol or air-dried. For microscopic study, they were prepared according to the protocol described by Breedy and Guzman (2002) , and observed using optic microscopy, Olympus LX 51 inverted microscope, and scanning electron microscopy, with a Hitachi 3700 at the

Research Center of Microscopic Structures

(CIEMIC) of the

University of Costa Rica

(UCR) and a Zeiss EVO 40 at the Electron Microscopy Laboratory (Tupper Research and Conference Center). The holotype and paratypes are deposited in the

Museo de Zoología, Universidad de Costa Rica

(MZUCR).
The taxonomic approach was by the evaluation of characters following Breedy and Guzman (2015 , 2016 ). Morphological characters of colonies and sclerites are presented PageBreakin Tables 1–2 and comparison with the type material of the related taxa in the genus. Measurements of branches are given taking in account the length of the calyces whether preserved in ethanol or dry. Terminology used in descriptions mostly follows Bayer et al. (1983) and Breedy and Guzman (2015 , 2016 ).

The powder (untreated) and the marc of the plant samples following the different extraction processes were prepared for scanning electron microscopy. For this, the herb powder was dried for at least two hours under vacuum between 40°-50 °C. The samples were sputtered, coated with gold and scanned at higher vacuum mode for any structural changes under an electron microscope (Zeiss EVO40) [24] , [25] (link).

The morphological features of synthesized DSE-AgNPs were characterized by SEM (Carl Zeiss EVO 40, Germany) with accelerating voltage of 20 kV. AgNPs were sonicated for 10 min before being used. The shape and size of AgNPs were characterized by higher resolution transmission electron microscope (HR-TEM). For HR-TEM, a drop of dispersed solution was placed on a copper grid at room temperature. The HR-TEM images were obtained using a Tecnai G2 (FEI, Electron Optics, USA) transmission electron microscopy with an accelerated voltage of 200 kV.

The morphology of samples was observed using a scanning electron microscopy (EVO40, Zeiss, Jena, Germany), at acceleration voltage of 18 kV. Before testing, all the specimens were sputter-coated with gold for 5 s using a Balzers coater (PV205P, Oerlikon Balzers Coating SA, Brügg, Switzerland).

The surface morphology of S. aureus treated with wild-type cryptdin-2 and its PEGylated variant was studied by Scanning Electron Microscopy (SEM) analysis^{39 (link)}. Briefly, S. aureus cells were grown to mid-logarithmic phase, washed and resuspended in PBS. The recombinant peptide variants (2 × MIC) were added separately to the tubes containing 10⁸ cells/mL and incubated for 1 h at 37 °C. Post treatment, cells were washed with PBS and fixed with Karnowsky´s fixative. Subsequently, the samples were dehydrated using ethanol and were imaged by Scanning Electron Microscope (Zeiss EVO40).