Low cost antiviral activity of Plodia interpunctella haemolymph in vivo demonstrated by dose dependent infection.

Given the ubiquity of infectious disease it is important to understand the way in which hosts defend themselves and any costs that they may pay for this defence. Despite this, we know relatively little about insect immune responses to viruses when compared to their well-characterized responses to other pathogens. In particular it is unclear whether there is significant haemocoelic response to viral infection. Here we directly examine this question by examining whether there is a dose-dependency in infection risk when a DNA virus is injected directly into the haemocoel. Infection from direct injection into the haemocoel showed a clear dose dependency that is indicative of an active intrahaemocoelic immune response to DNA viruses in insects. In contrast to the natural oral infection route, we found no measurable sublethal effects in the survivors from direct injection. This suggests that the immune responses in the haemocoel are less costly than those that occur earlier.

Examining the relationship between hemolymph phenoloxidase and resistance to a DNA virus, Plodia interpunctella granulosis virus (PiGV).

We have a detailed understanding of invertebrate immune responses to bacteria and fungal pathogens, but we know less about how insects respond to virus challenge. Phenoloxidase (PO) functions as an important immune response against many parasites and pathogens and is routinely used as a measure of immune competance. We examine the role of haemolymph PO activity in Plodia interpuncetella's response to its natural granulosis virus (PiGV). Larvae were challenged with virus by both oral inoculation of occluded virus (the natural infection route) and direct intrahaemocoelic injection of budded virus. Haemolymph was collected at time points post-viral challenge using a novel method that allows the volume of haemolymph to be quanitified. The haemolmyph was collected without killing the larvae so that haemolymph samples from individuals that developed viral disease could be distinguished from samples collected from those that fought off infection. The level of haemolymph PO activity in resistant larvae did not differ from control larvae. Therefore we have no evidence that PO is involved in resistance to virus in the haemocoel whether larvae are challenged naturally by oral innoculation or directly by intraheamocoelic injection. Phenoloxidase may therefore not be a relevant metric of immunocompetence for viral infection.

Sensitivity to bites by the bedbug, Cimex lectularius.

Bedbugs are a public health problem and can cause significant economic losses, but little is known about the effects of bites on humans. We reviewed case reports and published papers on bedbug bites to assess the empirical basis of the commonly cited figure that only approximately 80% of the population are sensitive to bedbug bites. We found the sensitivity estimate to be based on only one study carried out 80 years ago. However, this study did not account for the now well-established fact that only repeated exposure to external allergens leads to skin reactions. In our sample, 18 of 19 persons showed a skin reaction after bedbug exposure, but in most cases only after repeated controlled exposure. With repeated exposure, the latency between bite and skin reactions decreased from approximately 10 days to a few seconds. Our results are relevant for the hospitality industry, where apparently increasing infestation rates are likely to lead to an increase in the number of tourists and hotel employees exposed to bedbugs. Medical and public health professionals may expect to see an increase in the prevalence of people with bedbug bite sensitivity. The significance of the delayed reaction time of skin to bites may also have implications in litigation cases where people seek compensation.

Trauma, disease and collateral damage: conflict in cimicids.

The bed bugs and bat bugs (Hemiptera: Cimicidae) are unusual in being a gonochorist (separate male and female genders) taxon with obligate traumatic insemination. Males of all the species in this family have a lanceolate paramere (intromittent organ) which they use to pierce the female's body wall and inseminate directly into her haemocoel, despite the presence of a functional female genital tract. Mating is tightly linked to the feeding cycle in Cimex lectularius, the common bed bug. In this paper, I examine key aspects of the reproductive anatomy and behaviour of C. lectularius that underpin the nature of the conflict over mating rate in this species. I then examine the consequences of traumatic insemination for female fitness and examine potential mechanisms that might underpin those costs. Finally, the collateral consequences of the male reproductive tactic on other males of C. lectularius and the African bat bug, Afrocimex constrictus are examined.

Modulation of sexual signalling by immune challenged male mealworm beetles (Tenebrio molitor, L.): evidence for terminal investment and dishonesty.

Organisms partition resources into life-history traits in order to maximise fitness over their expected lifespan. For the males of many species fitness is determined by qualitative and quantitative aspects of costly sexual signals: The notion that epigamic traits are costly forms the cornerstone of those theories that propose parasites drive sexual selection. Consequently studies examining this notion assume sexual signalling is honest (i.e. driven by cost) when they seek to identify correlations or causal links between male immune function and attractiveness. We demonstrate that immune challenged males of the mealworm beetle, Tenebrio molitor, increased their investment in epigamic pheromone signals: these males became significantly more attractive to females whilst increasing the activity of a key immune effector system. In other words males increase terminal reproductive effort (invest in attractiveness) in response to a survival threat (immune insult). Consequently the signal preferred by the female is dishonest when considering the male's condition.

Immune function responds to selection for cuticular colour in Tenebrio molitor.

Cuticular colour in the mealworm beetle (Tenebrio molitor) is a quantitative trait, varying from tan to black. Population level variation in cuticular colour has been linked to pathogen resistance in this species and in several other insects: darker individuals are more resistant to pathogens. Given that cuticular colour has a heritable component, we have taken an experimental evolution approach: we selected 10 lines for black and 10 lines for tan adult cuticular phenotypes over at least six generations and measured the correlated responses to selection in a range of immune effector systems. Our results show that two immune parameters related to resistance (haemocyte density and pre-immune challenge activity of phenoloxidase (PO)) were significantly higher in selection lines of black beetles compared to tan lines. This may help to explain increased resistance to pathogens in darker individuals. Cuticular colour is dependent upon melanin production, which requires the enzyme PO that is present in its inactive form inside haemocytes. Thus, the observed correlated response to selection upon cuticular colour and immune variables probably results from these traits' shared dependence on melanin production.

Examining costs of induced and constitutive immune investment in Tenebrio molitor.

Central to the conceptual basis of ecological immunity is the notion that immune effector systems are costly to produce, run, and/or maintain. Using the mealworm beetle, Tenebrio molitor, as a model we investigated two aspects of the costs of innate immunity. We conducted an experiment designed to identify the cost of an induced immune response, and the cost of constitutive investment in immunity, as well as potential interactions. The immune traits under consideration were the encapsulation response and prophylactic cuticular melanization, which are mechanistically linked by the melanin-producing phenoloxidase cascade. If immunity is costly, we predicted reduced longevity and/or fecundity as a consequence of investment in either immune trait. We found a measurable longevity cost associated with producing an inducible immune response (encapsulation). In contrast to other studies, this cost was expressed under ad libitum feeding conditions. We found no measurable costs for constitutive investment in immunity (prophylactic investment in cuticular colour).

Invertebrate ecological immunology.

Ecological immunology is a rapidly expanding field that examines the causes and consequences of variation in immune function in the context of evolution and of ecology. Millions of invertebrate species rely solely on innate immunity, compared with only 45,000 vertebrate species that rely additionally on an acquired immune system. Despite this difference in diversity, most studies of ecological immunology focus on vertebrates. Here we review recent progress derived largely from the mechanistic analysis of invertebrate innate immunity. Using this empirical base, we pose general questions in areas that are of central importance for the development of ecological immunology.

Traumatic insemination and sexual conflict in the bed bug Cimex lectularius.

The bed bug, Cimex lectularius, has a unique mode of copulation termed "traumatic" insemination [Carayon, J. (1966) in Monograph of the Cimicidae, ed. Usinger, R. (Entomol. Soc. Am., Philadelphia), pp. 81-167] during which the male pierces the female's abdominal wall with his external genitalia and inseminates into her body cavity [Carayon, J. (1966) in Monograph of the Cimicidae, ed. Usinger, R. (Entomol. Soc. Am., Philadelphia), pp. 81-167]. Under controlled natural conditions, traumatic insemination was frequent and temporally restricted. We show for the first time, to our knowledge, that traumatic insemination results in (i) last-male sperm precedence, (ii) suboptimal remating frequencies for the maintenance of female fertility, and (iii) reduced longevity and reproductive success in females. Experimental females did not receive indirect benefits from multiple mating. We conclude that traumatic insemination is probably a coercive male copulatory strategy that results in a sexual conflict of interests.

Male calling song provides a reliable signal of immune function in a cricket.

A critical prediction of the immunocompetence handicap hypothesis is that the expression of secondary sexual traits should be positively correlated with pathogen resistance ability This correlation is necessary if females are to be able to use a particular sexual trait as an indicator of a male's resistance ability. In this study we document a positive correlation between a sexually selected component of the calling song of male house crickets (the number of syllables per chirp) and haemocyte load, an important determinant of the ability to encapsulate pathogens in insects. The results indicate that, by favouring males which produce more syllables per chirp, females may also select males with higher pathogen resistance ability, potentially generating either direct or indirect selection on female mating preferences.

Density-dependent prophylaxis in the mealworm beetle Tenebrio molitor L. (Coleoptera: Tenebrionidae): cuticular melanization is an indicator of investment in immunity.

If there are costs involved with the maintenance of pathogen resistance, then higher investment in this trait is expected when the risk of pathogenesis is high. One situation in which the risk of pathogenesis is elevated is at increased conspecific density. This paper reports the results of a study of density-dependent polyphenism in pathogen resistance and immune function in the mealworm beetle Tenebrio molitor. Beetles reared at high larval densities showed lower mortality when exposed to a generalist entomopathogenic fungus and a higher degree of cuticular melanization than those reared solitarily. The degree of cuticular melanization was a strong indicator of resistance, with darker beetles being more resistant than lighter ones regardless of rearing density. No differences were found between rearing densities in the levels of phenoloxidase, an enzyme key to the insect immune response. The results show that pathogen resistance is phenotypically plastic in T. molitor, suggesting that the maintenance of this trait is costly.

A mechanistic link between parasite resistance and expression of a sexually selected trait in a damselfly.

This paper examines a field-based insect system in which a signal trait and an immune effector system responsible for parasite resistance rely on the same melanin-producing enzyme cascade (phenoloxidase, PO). Observations and experiments on males of the calopterygid damselfly Calopteryx splendens xanthostoma revealed that resistance to the prevalent parasite in the study system (a eugregarine protozoan infecting the mid-gut) was correlated with quantitative aspects of the sexually dimorphic melanized wingspot of males, a trait that is produced and fixed before the host comes into contact with the sporozsoites of the parasite. Regulation of PO during experimental immune challenge showed that males with dark, homogenous melanin distribution in their wings showed no change in PO levels 24 h after challenge. By contrast males with lighter and/or more heterogenous melanin distribution in their wings tended to show higher PO levels 2-h after immune challenge. The changes in PO levels occur despite the lack of a relationship between wing-pigment distribution and the cellular encapsulation response. These results suggest a shared, limiting resource may form the mechanistic basis of the trade-off between a condition-dependent signal trait and immune function in this system.

Last male sperm precedence in a damselfly demonstrated by RAPD profiling.

We used the random amplified polymorphic DNA technique to determine last male sperm precedence (P2) in the damselfly Calopteryx splendens xanthostoma (Charpentier). We amplified DNA from mothers, putative fathers and from the embryos of individual offspring, and subsequently calculated band-matching coefficients between known first-order relatives (offspring within a clutch) and non-relatives (mothers and fathers) to estimate last-male paternity. The data indicate that, as in other Calopterygidae, P2 is high (0.98) in the bout of oviposition immediately following copulation, despite the fact that the males of this species do not completely remove the sperm of previous males (Siva-Jothy & Hooper 1995).

Unravelling the components that underlie insect reproductive traits using a simple molecular approach.

Applications of RAPD technology to questions of paternity and maternity in studies of insect reproductive traits are discussed. We present three case studies where RAPD fingerprinting reveals levels of complexity in insect systems that help us to understand the causal mechanisms underlying observed behaviour. Finally, we consider ways in which RAPD data can be analysed to generate information about kinship.