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

Dissection station and specimen preparation

After continuous blood-feeding for 24 h to 14 days, fleas were collected and sequentially surface washed with 10 % bleach (sodium hypochlorite) for 2 min, 70 % ethanol for 2 min, and 3 × sterile distilled water for 2 min to remove surface debris, dried flea feces, host blood, and microorganisms. Next, fleas were immobilized on ice (approximately 30 min). Aliquots of 15 µL of 0.01 M sterile phosphate-buffered saline (PBS) pH 7.2 were placed in the wells of a glass depression slide under a stereomicroscope. Immobilized individual fleas were then laid down on their side into the drop of PBS using fine-tipped forceps. For flea dissection, a sharp needle (27 G) attached to a 1 mL slip-tip syringe was held in each hand. A very fine needle (30 G) attached to a syringe and a dissection probe (microneedles/minuten nadeln mounted to a wooden applicator stick) were necessary for tissue manipulation (Fig. 1B). The materials and tools used for flea dissection are listed in Table S1. The manufacturer/supplier with a corresponding identifier is provided for each item. All essential steps of a flea dissection are thoroughly described in the following Section 2.3-2.4, and File S3.

Note: Fine-tipped forceps can be cleaned by rinsing in 10 % bleach or 70 % ethanol and dried with Kimwipes disposable wipers between different sample groups. Surface cleansing and recycling of needles should be avoided for safety reasons and hazard of contamination. If needles are compromised during the dissection process, we recommend replacing them with a new item.

Instruction for salivary glands dissection

Using the needle held in one hand, the flea should be gripped by the head capsule and securely pressed against the slide. Next, a small incision is made between the mesonotum and metanotum with the needle held in the other hand, and the exoskeleton is gradually separated into two halves (Fig. 2A-B). To remove the salivary glands from the body cavity, the posterior end of the abdomen is pulled away gently and very slowly from the head (Fig. 2C). The paired salivary glands, still attached with thin ducts, should appear as translucent ovoid-shaped organs (Fig. 2D). Lastly, clip the salivary ducts with a needle to detach the glands for tissue collection. In some instances, careful manipulation with a 30 G needle is necessary to separate the salivary glands from fat body cells or any connective tissue that remains attached to them.

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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.

Note: The critical part is pulling the abdomen away from the head. If the salivary ducts are severed during the process, it is possible to find the broken glands amongst the surrounding organs; however, that may be of limited success and require dexterity with dissection tools.

Dissection of the alimentary canal and female reproductive organs

Once the exoskeleton is dissected into two parts, the alimentary canal is freed to be isolated. If the oesophagus is broken (Fig. 2D), the digestive tract may be withdrawn from the abdomen as follows: the needle held in one hand is pressed firmly to the coxa and the midgut and hindgut are released from the abdomen by gently pulling the apex of the proventriculus with the needle held in the other hand (Fig. 2E-G). If the oesophagus is not severed during the procedure described in Section 2.3, the proventriculus, midgut, and hindgut should remain attached to the head (Fig. 2H).

After the alimentary canal is removed from the body cavity, the reproductive organs can be recovered intact. Firstly, the needle held in one hand is inserted inside the anterior end of the flea body and firmly pressed against the slide (Fig. 2I). Next, the chitin exoskeleton is nicked between the VI and VII tergum segments, and the posterior end is opened using the needle held in the other hand (Fig. 2J). While the sensillum is carefully pulled away from the rest of the abdomen, the ovarioles are dislodged and spread out on the slide (Fig. 2K-L). Lastly, the ovaries are dissected by slitting the tergum with the needle. The ovarioles can be debrided from the fat body using a dissection probe if necessary.

Note: After the dissection is completed, it is strongly recommended that the harvested flea tissues are rinsed with PBS (i.e., placing them to a new drop of PBS in the second well of the depression slide) before transferring biological material into a collection tube, in order to remove residual hematocytes, debris of exoskeleton, and undesired off-target tissues. Depending on the aim of the study, organs can be collected in RNAlater solution (for gene expression studies) or transferred directly to an extraction buffer, such as TRIzol Reagent (for nucleic acid isolation) or I-PER Reagent (for protein extraction). Alternatively, the dissected tissues may be stored in PBS at –80 °C.

Morphology of the cat flea salivary glands

Fleas have two pairs of salivary glands (four in total), two on each side of the abdomen, attached to a pair of salivary ducts. The two single cuticle-lined salivary ducts unite to form one long common tube (Fig. 3, Fig. 4). Representative photographs comparing salivary gland morphology of both sexes collected on different days post-feeding are shown on Fig. 3A. The maturation of cat flea gonads and oviposition results in changes of various internal organs, including the salivary glands, midgut epithelium, and fat body, which follow a developmental cycle. Specifically, in the female flea during oocyte maturation, the salivary gland cells greatly increase in size (Fig. 3A, top row; Fig. 4C), analogous to other members of the Pulicidae family: the rat flea Xenopsylla cheopis and the rabbit flea Spilopsyllus cuniculi (Rothschild et al., 1986).

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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.

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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.

Remarkably, among the numerous specimens studied, we spotted individuals with joined/fused and supernumerary salivary glands, while such abnormality was not sex-linked (Fig. 3B). Development and growth of insects is controlled by hereditary and environmental factors (e.g., nutrition and temperature). As a genetic factor, inbreeding may contribute to the emergence of morphological variation (Jong et al., 2017). In holometabolous insects, including the cat flea, tissue differentiation is connected to the growth and transcription factors that control metamorphosis within the pupa. Although organogenesis of model insect organisms is currently rather well understood (Nijhout et al., 2014; Texada et al., 2020; Nation 2022), the exact mechanisms that regulate the shape and size of different organs in the flea body remains unknown. Notably, it is unclear how phenotypic abnormalities recorded in our study affect the physiology and the secretory function of the salivary glands.

Architecture of cells constituting salivary glands

Using dyes with affinity to well-conserved organellar markers, we visualized the adult cat flea salivary glands by epifluorescent and confocal microscopy (Fig. 4). Employing an optimized protocol for fluorescent staining, we observed cellular characteristics of the glands. Cat fleas have reservoir-structure type glands, with a central lumen, surrounded by 8–10 secretory cells (Williams 1991; Ribeiro 1995). In agreement with previous reports, each gland consists of relatively large mono-nucleated cells, which create a unicellular epithelial layer around the bladder-like cavity. Applying Evans blue dye, we counterstained the permeabilized cells of glands, and using a higher magnification (i.e., 400 ×) we captured the striated cuticle of the duct (Fig. 4A). To obtain a view of the salivary glands` cytoskeletal architecture, we utilized a conjugated phalloidin probe for selective labeling of filamentous actin (Fig. 2B). The striking differences in the salivary glands structure during the course of blood-feeding and between sexes are highlighted in Fig. 4C.