Juvenile hormone metabolism in parasitized larvae of Lymantria dispar

Larvae of Lymantria dispar, parasitized by the endoparasitic wasp Glyptapanteles liparidis, show disruption of their development during their final host instar. From our previous studies we know that increased titers of juvenile hormones (JH) are involved in these changes resulting in developmental arrest and prevention of metamorphosis. Increased JH titers in parasitized larvae are caused at least in part by suppression of activity of juvenile hormone esterase (JHE), the enzyme responsible for degradation of JH in the hemolymph of L. dispar. Because of this crucial role of JHE in regulating the JH titer, the project focuses on the effects of parasitism on the expression and activity of this enzyme. During oviposition different associated factors such as venom, calyx fluid including polydnaviruses and also parasitoid eggs which release teratocytes (cells deriving from the serosa of the hatched parasitoid egg) are inserted into the host`s body. One aim of the project is to determine which of these components reduce activity of JHE. The second aim of the project is to determine the level on which suppression of JHE occurs. Thus, it is investigated whether parasitism interferes with the expression of the protein on the level of transcription or translation/posttranslation. Furthermore, a possible uptake into pericardial cells or an uptake by teratocytes resulting in a lower concentration of JHE in the hemolymph of parasitized larvae is examined.

The gregarious braconid Glyptapanteles liparidis is the dominant hymenopteran parasitoid of gypsy moth larvae in natural oak forests of eastern Austria and plays an important role in regulating the population densities of this episodic forest pest. Gypsy moth larvae parasitized by the endoparasitic wasp are induced to undergo developmental arrest before the parasitoids emerge. From previous studies we know that the juvenile hormone (JH) hemolymph titer of parasitized larvae is significantly higher than in unparasitized larvae. One role of high JH titres in parasitized larvae may be to maintain a larval-like nutritional milieu in the host which assures the parasites of access to hemolymph nutrients that might otherwise undergo sequestration. To investigate possible causes of the high JH levels in parasitized larvae, we studied to what extent individual factors associated with parasitism (venom and calyx, polydna virus, teratocytes, parasitoid larvae), contributed to low activity of hemolymph JH specific esterase (JHE), the only significant JH degrading enzyme present in hemolymph. First, we tested if possibly the parasitoids were able to regulate JHE enzyme activity by manipulating the host`s gene transcription which takes place in the fat body. We used real time PCR with gene specific primers based on the nucleotide sequence of a partial cDNA clone from gypsy moth JHE to determine the level of gene expression in 4th instar larvae. Parasitism had no influence on JHE gene activity; transcription levels were similar in both unparasitized and parasitized gypsy moth larvae. JHE expression was highest on day 1 of 4th instar and gradually decreased until day 5. On day 7, gene expression increased again. However, measurement of JHE enzyme activity from hemolymph of parasitized and unparasitized larvae showed that enzyme turnover was significantly lower in parasitized larvae throughout the 4th instar and remained extremely low even after parasitoids had left their host. These results and experiments with hemolymph injections from parasitized larvae made it obvious that neither gene expression nor a humoral factor were responsible for the suppressed JHE activity after parasitism. Thus, we still do not know if the inhibitory effects of parasitism on JHE activity are due to posttranscriptionalranslational regulation, resulting in low amounts of protein synthesized in parasitized larvae. Furthermore, we could demonstrate that in larvae parasitized with gamma radiated wasps (without viable eggs), esterase levels were lower than in untreated controls indicating the supporting activity of venom and polydna virus (PDVs) which are injected by the female at the time of parasitism. Teratocytes which we obtained from embryonic serosal cells after parasitoid hatch and that were injected into unparasitized hosts also inhibited the JHE activity. Second instar parasitoids implanted into control larvae suppressed the enzyme activity, whereas implanted 1st instar parasitoids had little effect on JHE. PDVs and venom injections cause host immune-suppression which allowed the implanted parasitoids to develop and moult, but not to emerge from the host. When parasitoids were implanted into untreated host larvae, they evoked instantly a strong host immune response by encapsulating the parasitoid larvae with hemocytes. The results of the different experiments show clearly that a concerted action of the many factors associated with parasitism is indispensable to take over control of the host`s endocrine and immune system and to allow parasitoids to develop successfully.