Evidence for the existence of juvenile hormones in ticks.

Abstract

Juvenile hormones (JHs) comprise a family of sesquiterpenoid compounds identified primarily from insects. JHs are involved in preventing metamorphosis during moults in immature stages and regulating aspects of reproduction in adults[l]. The JH titre is strictly regulated by a number of different mechanisms at specific times during development. JHs are synthesized and released into the haemolymph, where they are bound to haemolymph binding-proteins which protect them from esterases[2]. When JHs are no longer needed, they are degraded by specific JH-esterases and JH-epoxidases to the inactive products, JH-acids and JH-diols[3]. Ticks (Acarina:lxodidea), like insects, pass through several immature stages before becoming reproductively competent. JHs have not been identified in ticks, but several reports suggest that JHs are present and have roles similar to those in insects[4,5]. This study presents evidence for the occurrence of haemolymph JH-binding proteins and of specific JH-esterase and epoxidase activities, thus, indirectly supporting the presence of JH-like compounds in the adult tick, Amblyomma hebraeum. Ticks were raised as previously described[6] and taken for assay at various times during the feeding cycle. For JHesterase and epoxidase studies, tick tissues were divided into three categories: haemolymph, gut tissue and 'remaining tissues' (included fat body, muscle, ovary, salivary glands, and synganglion). Total esterase and epoxidase activities were determined by homogenizing the tissue in phosphate buffered saline (0.1M phosphate buffer, 100 mM NaCI) and incubating an aliquot with 0.12 HCi [3H]JHIII (0.58 Ci/mol) for 4h at 26'C. The sample was then extracted into acetonitrile and radioactivity analyzed by HPLC connected to an on-line radioactivity detector. Separation was performed on a C I S column using a linear gradient of acetonitri1e:water (35:65. v/v) to acetonitrile over 45 min at a flow rate of 1 ml/min. Non-specific esterase activity was assessed according to Rotin et a1.[7]. Haemolymph binding studies using the photoaffinity label, [3H]epoxy-bis-farnesyI diazoacetate ([3H]EBDA), followed the procedure of King ef al.[8]. For this. diluted haemolymph was incubated with 1 pCi [3H]EBDH or I 1.11 [3H]EBDA + 10 nmol JHI in polyethylene glycol-treated quartz cuvettes and exposed to 254 nm light for 5 min in a Rayonete Photoreactor. Following SDS-PAGE, gels were prepared for fluorography according to Prestwich et al. [9]. Incubation of tissues with [3H]JHIII suggests that only the 'remaining tissues' group showed specific esterase activity (total esterase activity non-specific esterase activity). Epoxidase and JH-specific esterase activities in this group of tissues were low in small, partially fed females, peaked just prior to engorgement. then declined to initial levels at the time of engorgement. Of the two enzymes examined, the JHepoxidase showed the greatest activity. Gut tissue apparently could only non-specifically metabolize JHIIl to the inactive -acid, -diol, and -acid-diol derivatives. Haemolymph tissue showed no detectable esterase or hydrolase activity but this could be due to difficulties in obtaining a sufficient volume of haemolymph for the assay. Photoaffinity labelling experiments using the JHI photoaffinity analogue, [3H]EBDA, with haemolymph from partially fed females indicated the presence of two regions of [3H]EBDA binding on SDS-PAGE gels that could be displaced by excess JHI. These corresponded to a high molecular weight region (75-100 kDa) and a single, low molecular weight band (approximate molecular weight 44 kDa). Kulcsar et al.[10] identified a similar, high molecular weight binding region (66-97 kDa) using the JHIII-specific photoaffinity label [3H]epoxy-farnesyl diazoacetate but suggested that this labelling was due to non-specific binding of [3H]EBDA to lipoproteins. It is likely that the high molecular weight region we observe is also due to nonspecific binding of lipoproteins. The 44 kDa labelled protein observed on SDS-PAGE gels is more suggestive of a JHIdisplaceable binding protein, however, at present i t is not known whether this protein is specific for a JH-like compound or is developmentally regulated throughout the feeding cycle. Although direct identification of JHs in tick haemolymph/tissue is ultimately needed, a combination of indirect evidence using complementary techniques can be strongly suggestive of the presence of JH. The identification of a haemolymph JHI-displaceable binding protein using the photoaffinity label EBDA indicates that a mechanism for JH protection and distribution exists in ticks. However, binding displacement by JHI does not imply that endogenous JHI is present in ticks and may merely reflect cross-reactivity to another related compound. Furthermore. the enzymes responsible for JH degradation in insects are also present in tick tissues and these enzymes appear to be developmentally regulated. Given that ticks possess these basic mechanisms for strict JH titre regulation, it is compelling to suggest that JHs do have important functions in tick development.