Insect Immunity to Parasites: Understanding Pathogen Defense Mechanisms

1. Transmission by Insect vectors • Insect ingested (double pored tapeworm) • As insect bloodfeeds, parasites in the salivary glands injected into the host along with the saliva (Plasmodium, Arboviruses). • As insect bloodfeeds, parasites in the mouthparts recognize the host as suitable and forcibly exit the mouthparts and enter the host (filarial nematodes). • As insect feeds in engorges and defecates, parasites in feces enter host (Chagas disease) • As insect feeds it regurgitates compounds from its crop and the parasites enter the host (Plague bacterium) • Parasites in 3 and 4 remain in the GI tract and do not come in contact with the tissues that are important in the invertebrate immune response to these parasites. • For the others…. Why do the insects tolerate the presence of the parasites? • Why do they not kill them? • How might they kill these parasites?

2. * reduced nutrients available to host * reduced synthesis of vitellogenin in fat body (Hogg et al. 1997) * ovary uptake of vitellogenin is impaired (Hogg et al. 1997) * increase in hemolymph yolk proteins * resorption of developing follicles (Carwardine and Hurd 1997) * reduced fecundity (Ahmed et al. 1999) * reduced fertility (Hacker 1971, Hogg and Hurd 1995) * bloodfeeding behavior affected (Anderson et al. 1999) Why should vectors protect themselves from parasites? Do human parasites affect their vectors? Fitness Costs Associated with Parasite Infection

3. The Vector-Parasite Relationship • Why do insects tolerate parasites and pathogens? • How do insects protect themselves against parasites and pathogens? • Why do insects not kill all parasites and pathogens? 3

4. I Why do the insects not kill their parasites??? To answer this we first must understand how insects CAN protect themselves, and then determine why these protective measures are not used or are not effective against the pathogens

5. INNATE IMMUNITY OF INSECTS Innate immunity refers to a nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to a stimulus. This is the immunity one is born with and is the initial response by the body to eliminate microbes and prevent infection. Unlike adaptive immunity, innate immunity does not recognize every possible antigen. Instead, it is designed to recognize a few highly conserved structures present in many different microorganisms. The structures recognized are called pathogen-associated molecular patterns (PAMPs). Most defense cells have pattern-recognition receptors for these common pathogen-associated molecular patterns to allow for an immediate response against invading microorganisms. Pathogen-associated molecular patterns can also be recognized by a series of soluble pattern-recognition receptors in the blood that function as opsonins and initiate the complement pathways. Examples of innate immunity include anatomical barriers, mechanical removal, bacterial antagonism, pattern-recognition receptors, antigen-nonspecific defense chemicals, the complement pathways, phagocytosis, inflammation, and fever.

6. pathogen-associated molecular patterns (PAMPs): conserved molecular patterns on microbes lipopolysaccharide (LPS) from the gram-negative bacteria cell wall; peptidoglycans found abundantly in the gram-positive cell wall and to a lesser degree in the gram-negative cell wall lipoteichoic acids found in the gram-positive cell wall; mannose-rich glycans (common in microbial glycoproteins and glycolipids); Β-glucans on fungi To recognize these microbial molecules, various body defense cells have on their surface a variety of receptors called pattern-recognition receptors capable of binding specifically to conserved portions of these molecules.

7. Pattern-Recognition Receptors (Including Toll-Like Receptors) • 1. Pattern Recognition Receptors (PRR ) • Recognize pathogen associated molecular patterns (PAMP); conserved molecular patterns on microbes • Toll-Like Receptors (TLR): • First discovered in Drosophila • Eleven receptors identified in mice and humans

8. Ligands are PAMP (pathogen- associated molecular patterns) Receptors are PRR (pattern- recognition receptors)

9. Nobel prize 2011

10. Immune Response of Insects Presence of Pathogens Recognition? Fat body Molecules of Molecules of communicacion communication Hemocytes? Serine Proteases ? Hemocytes Transferrin Activation via Toll, IMD, y IRD ProPO Serine Proteases Production of Tyrosine PO Immune peptides Phagocytosis DDC Antimicrobial compounds Defensins DCE Cecropins Proline-Rich Peptides Glycine-Rich Peptides Others? Antimicrobial Melanotic encapsulation Activity

11. E. coli 3 hr: Phagocytosis

12. Immune Response of Insects Presence of Pathogens Recognition? Fat body Molecules of Molecules of communicacion communication Hemocytes? Serine Proteases ? Hemocytes Transferrin Activation via Toll, IMD, y IRD ProPO Serine Proteases Production of Tyrosine PO Immune peptides Phagocytosis DDC Antimicrobial compounds Defensins DCE Cecropins Proline-Rich Peptides Glycine-Rich Peptides Others? Antimicrobial Melanotic encapsulation Activity

13. Melanotic encapsulation

14. Immune Response of Insects Presence of Pathogens Recognition? Fat body Molecules of Molecules of communicacion communication Hemocytes? Serine Proteases ? Hemocytes Transferrin Activation via Toll, IMD, y IRD ProPO Serine Proteases Production of Tyrosine PO Immune peptides Phagocytosis DDC Antimicrobial compounds Defensins DCE Cecropins Proline-Rich Peptides Glycine-Rich Peptides Others? Antimicrobial Melanotic encapsulation Activity

15. defensin gambicin Mosquito Antimicrobial Peptides cecropin lysozyme Bomanin Jacob

16. Advantages of Insect Innate Immune System * Antimicrobial peptide generation is very fast * Peptides are potent with wide spectrum of activity * Small peptides diffuse quickly * Insect immune peptides do not need special cells for production

17. Where do peptides act? How can parasites survive? • The majority of insect immune peptides are expressed in hemocytes and fat body tissues and secreted into the hemolymph • Parasites can: • Evade immune response • Inactivate immune response • Avoid contact with immune response

18. defensin defensin

19. DENv comprise 4 antigenically distinct serotypes: DENv-1, -2, -3, -4 2.5 billion people at risk 50-100 million new infections/year ~500,000 cases of DHF, DSS No vaccine, no drugs Transmitted by mosquitoes Aedes aegypti, Aedes albopictus, Aedes polynesiensis What happens with Intracellular Parasites? Intracellular viruses are not freely exposed to classical components of the vector immune response 19

20. Apoptosis: Programmed Cell Death Cellular response to damage, age, and stress Intracellular infection Cells respond to viral infection by initiating apoptotic cell death Powerful immune response severely limit virus production reduce or eliminate the spread of progeny virus 20

21. We believe: Dengue enters cells- Mosquito activates apoptosis virus over expresses IAP1 Apoptosis inhibited until virus has replicated Cells allowed to burst- releasing virions How can we prove/disprove/study this?

22. Cali, Colombia: • >120,000 dengue cases • 115 deaths • Naturally DENv resistant field population of Aedes aegypti 22

23. Costs associated with Parasitism: Reduced nutrients available to host Reduced synthesis of vitellogenin in the fat body Uptake of vitellogenin by the ovary is impaired Increased concentration of egg yolk proteins Resorption of developing follicles Reduced fecundity & fertility Bloodfeeding behaviour may be altered

24. Melanization * reduced fertility * increased time to oviposition * reduced longevity * competition for resources needed for egg * development and melanin synthesis Immune peptides and Phagocytosis * No apparent reduction in fertility * No significant reduction in longevity * Competition for resources? Fitness Costs Associated with Immune Response

25. Phenylalanine PAH Tyrosine Competition Defense Egg Development TRADE OFFS

26. Undergoing immune response Controls (Ferdig et al. 1993)

27. Why study insect immune responses? • Understand general insect immunity: the innate responses that insects use to protect themselves from pathogens • By understanding how this system works we may exploit it to enhance the success of management strategies • Invertebrates have no antigen:antibody system, and lack a memory function. But their innate immune response is extremely similar to that of the vertebrate acute phase response. We can look at the origin and progenitor responses that have arisen through different evolutionary periods and which form the only immune response in invertebrates. • Identify several novel immune peptides that have potent antimicrobial activity. These have a broad spectrum of activity, and can be produced and released with no known toxicity to eukaryotic organisms.

28. Rhodnius prolixus Anopheles gambiae T. Cruzi P. falciparum

29. Armigeres subalbatus Brugia pahangi Brugia malayi killed develops