Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

Summary

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis.

Abstract

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.

Introduction

Malaria is a major public health threat causing almost 230 million infections and an estimated 409,000 deaths in 2019¹. The majority of deaths are in sub-Saharan Africa and are caused by the parasite Plasmodium falciparum, whose insect vector is Anopheles gambiae, the subject of this video demonstration. Although the numbers indicate a significant drop in annual death rate since the turn of the century (>300,000 fewer annual deaths), the promising decreases in disease rates observed from 2000 to 2015 are tapering, suggesting the need for new approaches to limiting disease transmission². Among promis....

Protocol

1. Preparation of solutions and tools

1. Preparation of dissection solution
   1. To prepare dissecting solution, add 2.5 mL of 100% ethanol to 7.5 mL of distilled H₂O in a 15 plastic tube. Invert the tube 3 times to mix.
      NOTE: This solution can be stored at room temperature for several weeks.
2. Preparation of 10x phosphate-buffered saline (PBS) stock
   1. To prepare 10x PBS stock, add 17.8 g Na₂HPO₄• 2H₂O, 2.4 g KH₂PO₄, 80 g NaCl, and 2 g KCl to 800 mL of deionized water. Mix with a stir bar on a stir plate until the solids have....

Results

Salivary glands are relatively easy to dissect from all stage 4 larvae. Male and female larvae can be distinguished at the late L4 larval stage by a red stripe along the dorsal thorax of females but not males (Figure 2). We also observe that antennal morphology is much more elaborate in male than in female L4 larvae (Figure 2), similar to the differences observed in this structure in adult mosquitoes. Along with the considerable overall growth during the L4 stag.......

Discussion

The protocol described herein was adapted from a Drosophila SG dissection protocol and an adult mosquito dissection protocol^14,15,16. However, most markers did not penetrate the basement membrane (data not shown) when using the adult dissection and SG staining methods. Adaptations of the adult protocol included dissecting the glands in a 25% EtOH solution, washing the glands with a combination of MeOH and glacial acetic acid, an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
KH2PO4	Millipore Sigma	P9791
Na2HPO4 • 2H2O	Millipore Sigma	71643
NaCl	Millipore Sigma	S7653
Acetone	Millipore Sigma	179124
Brush with soft bristles	Amazon (SN NJDF) Detail Paint Brush Set	B08LH63D89
Cover slips (22 x 50 mm)	VWR	48393-195
DAPI (DNA)	ThermoFisher Scientific	D1306
Ethyl alcohol 200 proof	Millipore Sigma	EX0276
Gilson Pipetman P200 Pipette	Gilson	P200
Glacial Acetic Acid	Sigma Aldrich	695092
Jewelers forceps, Dumont No. 5	Millipore Sigma	F6521
KCl	Millipore Sigma	58221
Methanol	Millipore Sigma	1414209
Nail polish	Amazon (Sally Hansen)	B08148YH9M
Nile Red (lipid)	ThermoFisher Scientific	N1142
Paper towels/wipes	ULINE	S-7128
Petri plate (to make putty plate)	ThermoFisher Scientific	FB0875712
Pipette Tips	Gilson	Tips E200ST
Plastic Transfer Pipette	Fisher Scientific	S304671
Primary antibodies (e.g., Crb, Rab11)	Developmental Studies Hybridoma Bank (DSHB); Andrew Lab	Mouse anti-Crb (Cq4) or Rabbit anti-Rab11
Secondary antibodies with fluorescent tags (e.g., Alexa Fluor 488 Goat-anti Rabbit)	ThermoFisher Scientific	A11008
Silicone resin and curing agent for putty plate	Dow Chemicals - Ximeter Silicone	PMX-200
Slides, frosted on one end for labelling	VWR  20 X 50 mm	48393-195
Wheat Germ Agglutinin	ThermoFisher Scientific	W834