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Review
. 2024 Apr 4:5:100080.
doi: 10.1016/j.cris.2024.100080. eCollection 2024.

Salivary glands of the cat flea, Ctenocephalides felis: Dissection and microscopy guide

Monika Danchenko  1 , Kevin R Macaluso  1
Affiliations
Review

Salivary glands of the cat flea, Ctenocephalides felis: Dissection and microscopy guide

Monika Danchenko et al. Curr Res Insect Sci. .

Abstract

Fleas are morphologically unique ectoparasites that are hardly mistaken for any other insect. Most flea species that feed on humans and their companion animals, including the cat flea (Ctenocephalides felis), have medical and veterinary importance. Besides facilitating blood acquisition, salivary biomolecules can modulate pathogen transmission. Thus, dissection of salivary glands is essential for comprehensive studies on disease vectors like the cat flea. Herein, we present the pictorial dissection protocol assisting future research targeting individual flea organs, for revealing their roles in vector competence and physiology. We provide a comprehensive guide, allowing researchers, even with limited practical experience, to successfully perform microdissection for collecting cat flea salivary glands. Furthermore, the protocol does not require expensive, sophisticated equipment and can be accomplished with routinely available tools. We illustrated expected results with morphological changes of salivary glands upon blood feeding as well as fluorescently stained these organs.

Keywords: Disease vector; Dissection protocol; Flea tissue; Internal anatomy; Light microscopy; Pictorial guide.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image, graphical abstract
Graphical abstract
Fig. 1
Fig. 1
Overview of sample preparation. (A) Lateral view of adult female and male cat flea (C. felis) and their anatomical diagram with key morphological features. Photographs were taken with a Moticam 1080 camera attached to a Motic SMZ-143 stereomicroscope. Scale bar represents 1 mm; (B) Dissection station and specimen preparation. The protocol details are given in Materials and Methods, Section 2.2. Abbreviation: PBS, phosphate-buffered saline, pH 7.2. Created with BioRender.com.
Fig. 2
Fig. 2
Step-by-step guide of flea dissection. The depicted specimen is an adult female collected 7-days post-feeding, undergoing active oviposition. The pictorial dissection walkthrough of the salivary glands (panels A-D), the alimentary canal (panels E-H), and female reproductive organs (panels I-L) is extensively described in Materials and Methods, Section 2.3-2.4. If the operator is left-handed, all displayed hand motions should be reversed. For the fine microdissection of a cat flea, a 27 G needle attached to a thin syringe is recommended. The dissection procedure was recorded via Moticam 1080 camera attached to a Motic SMZ-143 stereomicroscope, using a reflective light source, apart from panel H, acquired with a transmissive light source. The terminology of the flea's internal anatomy is according to Rothschild et al. (1986). Abbreviations: AG, abdominal ganglia of the ventral nerve cord; FB, fat body; MG, midgut; MT, Malpighian tubule; OE, oesophagus; OV, ovarioles; PV, proventriculus; SG, salivary glands. Created with BioRender.com.
Fig. 3
Fig. 3
Dissected cat flea salivary glands, reproductive tissues, and digestive tract observed with bright-field light microscopy. (A) Changing morphology of salivary glands through the course of blood-feeding. Differences were recorded between the sexes after 1-, 3-, 5-, 7-, 10-, and 14-days of continuous feeding (dpf). Scale bar represents 200 μm; (B) Variety of organ malformation and inter-individual variability of the flea's salivary gland morphology. Two types of tissue abnormality were observed: i) joined/fused glands, and ii) presence of an additional 5th gland. Scale bar represents 200 μm; (C) Maturation of female reproductive tissues and development of oocytes linked to blood acquisition, documented 24–72 h post-feeding (hpf). Scale bar represents 500 μm; (D) Digestive tract of the cat flea containing partially digested bloodmeal. Scale bar represents 200 μm. Micrographs displayed on panels A, B, and D were obtained using an Olympus DP72 camera attached to an MVX10 stereo dissecting microscope with a transmissive light source. Photographs on panel C were captured with a Moticam 1080 camera attached to a Motic SMZ-143 stereomicroscope using transmissive light. Created with BioRender.com.
Fig. 4
Fig. 4
Adult salivary gland cellular architecture of the cat flea (C. felis). (A) Fluorescent staining of female salivary glands dissected 3-days post-feeding. The tissues were fixed with Bouin's solution and permeabilized using 0.1 % Triton X-100 in PBS. Nuclei were labeled with DAPI (blue signal in merged images), and salivary glands were counterstained with Evans blue (red fluorescent signal in merged images). Scale bar represents 100 μm (upper row) and 50 μm (lower row); (B) Fluorescent staining of female salivary glands dissected 7-days post-feeding. The samples were fixed with 4 % paraformaldehyde in cytoskeleton-stabilizing PHEM buffer and permeabilized using 0.1 % Triton X-100 in PBS. Actin filaments were labeled with conjugated phalloidin (purple signal), and nuclei were stained with DAPI (blue signal). Salivary glands were treated with a Vector TrueVIEW autofluorescence quenching kit. Scale bar represents 100 μm; (C) Comparative images of female salivary glands 1- and 14-days post-feeding (dpf), and differences in salivary gland structure between sexes. The preparations were fixed with Bouin's solution and permeabilized with 0.1 % Triton X-100 in PBS. Gland and duct nuclei were visualized with DAPI (blue signal). Scale bar represents 100 μm. The terminology of the flea's internal anatomy is according to Rothschild et al. (1986). Abbreviations: BF, bright-field microscopy; CD, striated cuticle of duct; EC, epithelial (secretory) cell; L, lumen; N, nucleus; SD, salivary duct; SG, salivary glands. Micrographs on panel A were taken using a Nikon Eclipse 90i microscope, and images on panels B and C were captured using an LSM 980 Airy Scan II confocal microscope. Created with BioRender.com.
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