An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Summary

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Abstract

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.

The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.

Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts¹. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response². Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited¹. Strains of L. boulardi also produce virus-like particles³ although they differ significantly in their ability to succeed on D. melanogaster¹. Some of these L. boulardi strains are difficult to grow on D. melanogaster¹ as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.

In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms^1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.

Wasp infection activates immune signaling (Fig. 4)⁴. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)^5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).

We present representative data with Toll signal transduction pathway components Dorsal and Spätzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

Protocol

The entire protocol for the experiment is divided into four steps (Fig. 9). (1) Culturing wasps on fly larvae; (2) Setting up infections and preparing animals for dissection; (3) Isolating and fixing host/parasite structures; (4) Analyzing immune tissues.

1. Culturing Wasps on Drosophila Larvae

Maintenance of wasp cultures requires careful planning. Relative to growing flies, it is fairly labor intensive. We maintain our wasp colonies in the laboratory on larvae or pupae of the y w strain of Drosophila at 24 °C. Culturing wasps involves keeping a continuous source of host....

Discussion

Interest in parasitic wasps of Drosophila is surging as molecular techniques to decode whole genomes become efficient and cost-effective. However, relative to their exceptionally well-studied hosts, many fascinating aspects of wasp biology remain obscure. These include issues related to host range, immune suppression, superparasitism, and behavior. The focus of this presentation was to demonstrate the effects of infection on the fly's immune tissues. The dissection techniques demonstrated here can be used for th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Yeast	Fisher Scientific	S80245-3	Active Dry
Fly Food	Home made	Corn meal, sugar	Standard
Honey	Dutch Gold	From the store	Clover
Vials	Fisher Scientific	AS514
Cotton plug/Foam stopper	Fisher Scientific	14-127-40A
Spatula	Fisher Scientific	21-401-15
Pyrex dissecting dish	Fisher Scientific	50-930-382	9-well
Microscope slides pre-cleaned	Fisher Scientific	7101	25.4 mm x 76.2 mm
Glass coverslips	Pearl	D12544	22 x 50
Glass coverslips	Pearl	G12544	22 x 60
Pasteur Pipette	J H Berge	71-5200-05
100 mm Tweezers	Sigma-Aldrich	T-4662	Style # 5
Wash Bottle	Fisher Scientific	03-409-22A	Fisherbrand
Kim Wipess	Fisher Scientific	34155	Kimberly Clark
Paper Towel	Fisher Scientific	Fisher X-101-C	1/8 x 13 1/8 in.
Leica stereomicroscope	Empire Imaging Systems, INC.	10446208	MZFLIII
Zeiss Stereomicroscope	Carl Zeiss, Inc.	000000-1006-126	"Stemi" 1000 or 2000-C
Light Source -LED Gooseneck illuminator	Fisher Scientific	12563501
Stage	Carl Zeiss, Inc.
Zeiss Laser 510 Scanning Confocal Microscope	Carl Zeiss, Inc.
Incubator	Thermo Fisher Scientific, Inc.	815
Dulbecco’s Phosphate-Buffered Salt Solution 1X	Fisher Scientific	MT-21-030-CM Mediatech
Ethanol	Fisher Scientific	64-17-5	99.5 %
Formaldehyde (37% w/w)	Fisher Scientific	F79-1
H–chst 33258	Molecular Probes, Life Technologies	H-1398	0.2 μg/ml Excitation 352 nm; Emission 461 nm
Rhodamine phalloidin	Molecular Probes, Life Technologies	R415	200 units/ml (6.6 μM) Excitation 546 nm; Emission 565 nm
Alexa Fluor 488 phalloidin	Molecular Probes, Life Technologies	A22289	Excitation 495 nm; Emission 518 nm 300 units/ml
CO2 tank	TW Smith
Anti-Spz antibody	Gift Dr. C. Hashimoto (Yale)
Secondary antibody TRITC-conjugated donkey anti-rabbit, concentration 1:50.	Jackson ImmunoResearch	711-165-152	Detected at Excitation 546 nm; Emission 565 nm
Antifade	MP Biomedicals	ICN10274790	0.08 mg of N-propyl gallate in 20 ml of 50% glycerol in 1X PBS
Wasp Strains			Fly Strains
L. victoriae16			y w
L. boulardi 171			UAS-GFP-Dorsal17
L. heterotoma2			SerpentHemoGal413
L. heterotoma 141			MSNF9-moCherry14
Trichopria drosophilae			MSNF-GFP15
G. xanthopoda18			y w Serpent-Gal4 UAS GFP-Dorsal/Basc4
y w ; Drosomycin-GFP/CyO y+12