Transmission by Insect vectors Insect ingested double pored

Transmission by Insect vectors Insect ingested (double pored tapeworm) 1) As insect bloodfeeds, parasites in the salivary glands injected into the host along with the saliva (Plasmodium, Arboviruses). 2) As insect bloodfeeds, parasites in the mouthparts recognize the host as suitable and forcibly exit the mouthparts and enter the host (filarial nematodes). 3) As insect feeds in engorges and defecates, parasites in feces enter host (Chagas disease) 4) As insect feeds it regurgitates compounds from its crop and the parasites enter the host (Plague bacterium) 5) 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. 6) For the others…. Why do the insects tolerate the presence of the parasites? 7) Why do they not kill them? 8) How might they kill these parasites?

Why should vectors protect themselves from parasites? Do human parasites affect their vectors? Fitness Costs Associated with Parasite Infection * * * * 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)

The Vector-Parasite Relationship 1) Why do insects tolerate parasites and pathogens? 2) How do insects protect themselves against parasites and pathogens? 3) Why do insects not kill all parasites and pathogens? 3

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

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.

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.

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

Ligands are PAMP (pathogenassociated molecular patterns) Receptors are PRR (patternrecognition receptors)

Nobel prize 2011

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

E. coli 3 hr: Phagocytosis

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

Melanotic encapsulation

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

Mosquito Antimicrobial Peptides lysozyme Bomanin Jacob cecropin defensin gambicin

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

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: 1) Evade immune response 2) Inactivate immune response 3) Avoid contact with immune response

defensin

What happens with Intracellular Parasites? 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 Intracellular viruses are not freely exposed to classical components of the vector immune response 19

Apoptosis: Programmed Cell Death • Cellular response to damage, 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

We believe: Dengue enters cells- Mosquito activates apoptosis virus over expresses IAP 1 Apoptosis inhibited until virus has replicated Cells allowed to burst- releasing virions How can we prove/disprove/study this?

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

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

Fitness Costs Associated with Immune Response 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?

TRADE OFFS Phenylalanine PAH Tyrosine Competition Defense Egg Development

Undergoing immune response (Ferdig et al. 1993) Controls

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.

Rhodnius prolixus T. Cruzi Anopheles gambiae P. falciparum

Armigeres subalbatus Brugia pahangi develops Brugia malayi killed