Bioactive fractions containing methyl eugenol-derived sex pheromonal components in haemolymph of the male fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

Sex pheromonal components of the tephritid fruit fly Bactrocera dorsalis (Hendel), 2-allyl-4,5-dimethoxyphenol and (E)-coniferyl alcohol, are biosynthesized from a highly potent male attractant, methyl eugenol, then sequestered and stored in the rectal gland prior to their release during courtship at dusk. These sex pheromonal components have been detected in the haemolymph and crop organ. Hence, attempts were made to separate and identify the haemolymph fractions which contained the sex pheromonal components. Identification of these bioactive fractions in methyl eugenol-fed male flies using gel filtration column chromatography and biodetection using live male flies showed two fractions as highly attractive to conspecific males. These fractions show a significant increase in protein absorbance in the elution profile of haemolymph from methyl eugenol-fed males compared with that from methyl eugenol-deprived males. The molecular mass of these bioactive fractions as determined by using gel filtration was in the peptide range of 3.3 to 5.5 kDa. Subsequent gas chromatography-mass spectrometry analyses further confirmed the presence of the pheromonal components in the bioactive fractions. The presence of these methyl eugenol-derived sex pheromonal components in specific haemolymph fractions suggests the involvement of a sex pheromone binding complex.     

Comparison of methyl eugenol metabolites, mitochondrial COI, and rDNA sequences of Bactrocera philippinensis (Diptera: Tephritidae) with those of three other major pest species within the dorsalis complex

Males of the oriental fruit fly, Bactrocera dorsalis (Hendel) and some of its sibling species have strong affinity for methyl eugenol (ME). Methyl eugenol ingested by male flies is biotransformed in the crop to two ME metabolites that eventually accumulate in the rectal gland, which is known to serve as a reservoir for B. dorsalis sex pheromones. When fed with ME, males of laboratory and wild B. philippinensis Drew and Hancock selectively accumulated two metabolites, 2-allyl-4,5-dimethoxyphenol and (E)-coniferyl alcohol, in the rectal gland, as was seen for B. dorsalis sensu stricto, B. invadens Drew, Tsuruta and White, and B. papayae Drew and Hancock. Phylogenetic analysis of COI and rDNA sequence data of these four taxa also revealed a close relationship among B. philippinensis, B. dorsalis s.s., B. invadens, and B. papayae (all four are members of the dorsalis species complex). This result corroborates pheromone analysis. The usefulness of pheromonal analysis as a chemotaxonomy tool to complement molecular and other analysis in differentiation of closely related sibling species within the Bactrocera dorsalis complex, for which use of morphological characters had been inadequate, is highlighted.     

Methyl eugenol and cue-lure traps for suppression of male oriental fruit flies and melon flies (Diptera: Tephritidae) in Hawaii: effects of lure mixtures and weathering

Methyl eugenol (4-allyl-1,2-dimethoxybenzene-carboxylate) and cue-lure [4-(p-acetoxyphenyl)-2-butanone] are highly attractive kairomone lures to oriental fruit fly, Bactrocera dorsalis (Hendel), and melon fly, B. cucurbitae (Coquillett), respectively. Plastic bucket traps were evaluated as dispensers for methyl eugenol and cue-lure for suppression of the 2 fruit flies in Hawaii. Methyl eugenol and cue-lure mixtures were compared with pure methyl eugenol or cue-lure over 4 seasons. B. dorsalis captures differed significantly with treatment and season. B. dorsalis captures with 100% methyl-eugenol were significantly greater than all other treatments (25, 50, and 75%). B. cucurbitae captures also differed significantly with treatment but not with season. Captures with 100, 75, and 50% cue-lure were not significantly different. Bucket traps baited with cue-lure (+ malathion) and weathered under Hawaiian climatic conditions were attractive to B. cucurbitae up to 8 wk. Two methyl eugenol dispensers (canec disks and Min-U-Gel) were compared with bucket traps. Dispensers (methyl eugenol + malathion) were weathered for 2-16 wk under Hawaiian climatic conditions and bioassayed during summer and winter. Initially, captures of B. dorsalis were not significantly different for the 3 dispensers. Bucket traps and canec disks were most resistant to weather, remaining attractive to B. dorsalis flies up to 16 wk. Min-U-Gel was least resistant, losing attractiveness to B. dorsalis flies within 2 wk. On the basis of performance, bucket traps and canec disks were equally long-lived up to 14 wk; thereafter, bucket traps were slightly more attractive during winter. Canec disks were cheapest, but on the basis of possible environmental concerns, bucket traps may be the best all-around choice for areawide suppression of fruit flies.     

Feeding behaviour of Bactrocera cacuminata (Hering) (Diptera: Tephritidae) on methyl eugenol: a laboratory assay

Abstract Many dacine (Diptera: Tephritidae) species are attracted to one of two chemical substances, 4-allyl-1,2-dimethoxybenzene (methyl eugenol (ME)) or 4-( p -hydroxyphenyl)-2-butanone (cue lure). Despite the fact that these chemicals or analogs occur naturally, their significance in the biology/ecology of the Dacinae has seldom been examined. In this study, we examine the patterns in feeding behaviour of Bactrocera cacuminata (Hering), a monophagous non-pest fruit fly, on ME. Based on a laboratory assay, we found that males of this species fed on ME multiple times within a single day and on multiple days. The pattern of repeat feeding was not related to time since previous feeding or duration of prior feeding. Our results contrast those obtained on a similar study on a related polyphagous species ( Bactrocera dorsalis (Hendel)). We discuss the implications of our findings with a view to explaining the functional significance of dacine response to these plant-derived chemicals.     

Antioxidant properties of 8.O.4′-neolignans

A series of naturally occurring 8.O.4′-neolignans (1a–d, 1g, 2g, 2h) and their analogues (1e–f, 1h, 1i, 2a–f, 2i) have been synthesized in racemic form starting from commercially available phenols, such as eugenol, isoeugenol and 4-allyl-2,6-dimethoxyphenol and from aromatic aldehydes, such as piperonal, veratraldehyde and 3,4,5-trimethoxybenzaldehyde. The inhibitory activity of these compounds on superoxide anion (O₂^.-) release by human polymorphonuclear leukocytes (PMNLs) was tested and the structure-activity relationship was also studied.     

Polyunsaturated hydrocarbons in the hemolymph: biosynthetic precursors of epoxy pheromones of geometrid and arctiid moths

Female moths of many species in Geometridae, Arctiidae and some other macrolepidopteran families produce epoxy pheromones, which are probably derived from polyunsaturated hydrocarbons. In order to understand a biosynthetic site, hemolymph from both sexes of two geometrid species, Ascotis selenaria cretacea and Hemerophila artilineata, and one arctiid species, Spilosoma imparilis, was shaken with n-hexane and the solvent extracts were analyzed by GC-MS. Each extract of the female hemolymph sex-specifically included polyunsaturated hydrocarbons corresponding to the pheromonal epoxy components in addition to many saturated hydrocarbons, but no epoxy compounds were detected in it. Based on this analysis, deuterated polyunsaturated hydrocarbons were injected into the abdomens of two geometrid females, and the labeled epoxy components were successfully yielded from the pheromone glands. This result indicated that the polyunsaturated hydrocarbons occurring in the female hemolymph were direct pheromone precursors, which might be produced outside the pheromone gland, probably in oenocytes associated with abdominal epidermal cells or in the fat body, and transported to the pheromone gland via the hemolymph for their epoxydation and emission into the atmosphere.     

Field trials of spinosad as a replacement for naled, DDVP, and malathion in methyl eugenol and cue-lure bucket traps to attract and kill male oriental fruit flies and melon flies (Diptera: Tephritidae) in Hawaii

Spinosad was evaluated in Hawaii as a replacement for organophosphate insecticides (naled, dichlorvos [DDVP], and malathion) in methyl eugenol and cue-lure bucket traps to attract and kill oriental fruit fly, Bactrocera dorsalis Hendel, and melon fly, B. cucurbitae Coquillett, respectively. In the first and second methyl eugenol trials with B. dorsalis, naled was in the highest rated group for all evaluation periods (at 5, 10, 15, and 20 wk). Spinosad was equal to naled at 5 and 10 wk during both trials 1 and 2, and compared favorably with malathion during trial 2. During the first cue-lure trial with B. cucurbitae, naled and malathion were in the top rated group at 5, 10, 15, and 20 wk. Spinosad was equal to naled at 5 wk. During the second cue-lure trial, spinosad and naled were both in the top rated group at 10, 15, and 20 wk. Use of male lure traps with methyl eugenol or cue-lure had no effect on attraction of females into test areas. Our results suggest that spinosad, although not as persistent as naled or malathion, is safer to handle and a more environmentally friendly substitute for organophosphate insecticides in methyl eugenol and cue-lure traps for use in B. dorsalis and B. cucurbitae areawide integrated pest management programs in Hawaii.     

对性引诱剂监测桔小实蝇雄成虫技术的改进

研究了添加方式、监测点位置以及监测区域环境对应用性引诱剂甲基丁香酚监测桔小实蝇Bactrocera dorsalisHendel雄成虫动态效果的影响。试验结果表明,采用分期分批、多次添加性引诱剂的方式监测桔小实蝇雄成虫动态,可消除因监测区域内所有诱瓶同一时间添加性引诱剂而造成的浓度突变所引起的成虫出现干扰性高峰,获得更准确的虫情监测结果;监测点的设置位置明显影响监测结果,监测点应包括果园内和果园外;选择较大且环境复杂的区域作为监测点可获得更明确的年发生动态规律。在以上研究基础上,提出了应用性引诱剂监测桔小实蝇雄成虫动态的监测方法。     

Isolation and identification of terpenoid sex pheromone components from extracts of hemolymph of males of the Caribbean fruit fly

Extracts obtained from hemolymph of sexually mature males of the Caribbean fruit fly Anastrepha suspensa contained four biologically important terpenoid components of the sex pheromone. The four components were identified as farnesene, bisabolene, anastrephin, and epianastrepin based on their relative retention indexes from capillary gas chromatography analysis, using both apolar and polar phase columns and their chemical ionization (isobutane) mass spectra. The ratio of the components in extracts of hemolymph was the same as the ratio present in the volatile blend of pheromone released by sexually mature males during the reproductive period. Studies conducted to determine the effect of age on amounts of these components in hemolymph indicated that they increased from undetectable levels on the day of adult emergence to maximum levels on day eight. The increases in amounts of the components present in hemolymph with increasing age were correlated with increases in amounts of volatile pheromone released by males. Time of day studies showed that the amounts of these components in hemolymph followed the daily pattern of release of volatile pheromone components. Other components of the sex pheromone including ocimene, (Z)-3-nonen-1-ol, (Z,Z)-3,6-nonadien-1-ol and suspensolide were not found in extracts of hemolymph. The data suggest that the hemolymph plays a role in the transport of these pheromone components during sexual signalling. Arch. Insect Biochem. Physiol. 42:225-232, 1999. Published 1999 Wiley-Liss, Inc.     

广州桔小实蝇(Bactrocera dorsalis (Hendel))发生动态及气象因子

2002～2005年期间,在广东广州利用性引诱剂对桔小实蝇进行了全年种群动态监测,调查可知桔小实蝇可在广州全年发生.该虫数量从5月开始迅速上升,6～9月份是发生盛期;10月份虫口密度逐渐下降,11月到翌年3月份种群数量很低.对桔小实蝇发生数量和气象因子进行主成分分析和相关分析,结果表明温雨因子作用最大,其中月平均降雨量是影响桔小实蝇种群变动的关键因子;日照因子作用次之,但月总日照时数对该虫的发生数量没有显著影响.     

Inositol lipid turnover and adenosine 3,5 cyclic monophosphate in the salt-secreting rectal gland of the dogfish (Scyliorhinus canicula)

The rectal gland of the dogfish is rich in inositol lipids. Total phospholipids from the gland contained 9.1 mol% of phosphatidylinositol (PtdIns), 1.0 mol% of phosphatidylinositol 4-phosphate (PtdIns4P) and 0.9 mol% of phosphatidylinositol 4,5-biphosphate (PtdIns4,5P2). [32P]Orthophosphate was readily incorporated into PtdIns, phosphatidic acid (PtdA) and especially into PtdIns4P and PtdIns4,5P2 in salt gland slices incubated in elasmobranch Ringer with glucose and no other additions over a 2 hr period. The calcium ionophore A23187 stimulated incorporation into PtdIns and PtdA, but not into PtdIns4P or PtdIns4,5P2. Oxygen uptake by rectal gland slices was maximally stimulated by 0.08mM forskolin, 2.5mM 8-chlorophenylthio cyclic AMP, 2.0mM dibutyryl cyclic AMP and 0.25mM theophylline. Stimulated oxygen uptake was inhibited by 0.1mM ouabain in all cases. Incorporation of [32P]orthophosphate into PtdIns, PtdA, PtdIns4P and PtdIns4,5P2 was inhibited by 0.08mM forskolin and 2.0mM dibutyryl cyclic AMP over a 2 hr period. The results are discussed in relation to the control of salt secretion by the rectal gland.     

Chemical alarm and defence in the oribatid mite Collohmannia gigantea (Acari: Oribatida)

The multicomponent oil gland secretion of Collohmannia gigantea, a middle-derivative mixonomatan oribatid mite, is demonstrated to possess alarm pheromonal and allomonal properties. Four components of the secretion, namely the monoterpenes neryl formate, neral, geranial and the aromatic 2-hydroxy- 6-methyl-benzaldehyde (2,6-HMBD), showed moderate to strong alarm pheromonal activity in adult mites. Naturally elicited response is due to neral (about 50% of the secretion) and probably 2,6-HMBD (only 5% of the secretion, but strong alarm pheromonal activity). This is the second report of an alarm pheromone in Oribatida. Tridecane and pentadecane (=the hydrocarbon fraction of the secretion) did not evoke evident behavioural reactions, and most likely serve as solvents and spreading agents for the pheromonal-active components. Alarm reactions were characterized by a short recognition phase (waving movements with legs I), followed by shrinking back and panic escape from the scent source. In addition, all six components of the oil gland secretion, including the hydrocarbons, exhibited strong allomonal properties against a model oribatid predator, the scydmaenid beetle, Euconnus (Tetramelus) oblongus. Considering the widespread semiochemical properties of oil gland secretions in astigmatid mites (=a highly derivative oribatid group), these results furnish evidence for a phylogenetically early origin of defensive and communicative roles of oil gland secretions in oribatids. These roles include alarm communication, defence and the production of anti-fungal compounds.     

Transport of a hydrophobic biosynthetic precursor by lipophorin in the hemolymph of a geometrid female moth which secretes an epoxyalkenyl sex pheromone

Previous experiments with a geometrid species, Ascotis selenaria cretacea, have suggested that a pheromonal C19 3,4-epoxy-6,9-diene is biosynthesized from the corresponding 3,6,9-triene produced outside a pheromone gland and transported to it via hemolymph after association with lipophorin. In order to clarify this transport, high-density lipophorin (HDLp) in the female moths showing two bands (apoLp I with ca. 250 kDa and apoLp II with ca. 80 kDa) on an SDS-PAGE was purified by KBr equilibrium density-gradient ultracentrifugation, and the association of the triene was confirmed by GC-MS analysis of a solvent extract from the isolated protein. Next, the role of HDLp was revealed by a topical application of the deuterated trienyl precursor to the abdomens of the females. The trienyl precursor was associated with HDLp. In their pheromone glands, the triene and the deuterated epoxy pheromone were detected, indicating movement of the triene via the hemolymph. Experiments with male moths of A. s. cretacea and female moths of Bombyx mori showed the same association of HDLp with the triene topically applied. This result suggested that the adult females of A. s. cretacea did not develop HDLp specialized in the triene transport. Furthermore, the topical application of a mixture including the trienyl precursor and two other related hydrocarbons showed equal amounts of association by HDLp but selective delivery of the precursor to pheromone glands in the A. s. cretacea females.     

Functional and expression pattern analysis of chemosensory proteins expressed in antennae and pheromonal gland of Mamestra brassicae

Sequences coding for chemosensory proteins (CSP) CSPMbraA and CSPMbraB, soluble proteins of low mol. wt, have been amplified using polymerase chain reaction on antennal and pheromonal gland complementary DNAs. On the basis of their sequences, these proteins could be classed in the 'OS-D like' protein family whose first member was described in Drosophila, and that includes proteins characterized in chemosensory organs of many insect phylla, including our recent identification in Mamestra brassicae proboscis. Binding assays have shown that these proteins bind the pheromonal component (Z)-11-hexadecenyl-1-acetate (Z11-16:Ac) as well as (Z)-11-octadecenyl-1-acetate (Z11-18:Ac), an other putative component of the M. brassicae pheromonal blend. Furthermore, binding with fatty acids, but not with progesterone that is a structurally unrelated compound, leads to the hypothesis that the odorant-binding capability of the MbraCSPs may be restricted to fatty acids and/or to 16-18 carbon backbone skeletons. Thus, these proteins do not show the same highly binding specificity as the pheromone-binding proteins do. The CSP-related proteins appear homologous based on sequence identity, conserved cysteine residues and general patterns of expression. However, phylogenetic analyses suggest the presence of multiple classes of CSP within a given species and possible diversification of CSPs within different orders. This diversity perhaps contributes to the many CSP functions proposed in the literature. In M. brassicae, we localized the CSPMbraA expression to the sensilla trichodea, devoted to pheromone reception, suggesting a role in the chemosensory pathway. However, we also localized such proteins in the pheromonal gland, devoid of any chemosensory structure. This suggests that the M. brassicae CSP could be involved in transport of hydrophobic molecules through different aqueous media, such as the sensillar lymph, as well as the pheromonal gland cytosol.     

Condition-Dependent Mating Success in Male Fruit Flies: Ingestion of a Pheromone Precursor Compensates for a Low-Quality Diet

Previous studies revealed that males of the oriental fruit fly, Bactrocera dorsalis, require protein in the adult diet to obtain matings and that ingestion of methyl eugenol, which acts as a pheromone precursor, increases male attractiveness and mating competitiveness. The goal of this study was to investigate the interaction between diet quality and methyl eugenol consumption in affecting the mating frequency of B. dorsalis males. In one set of experiments, mature males were deprived of protein for 1, 3, or 7 days and were either given or denied access to methyl eugenol (ME). These males competed against control males (continuously protein-fed, no feeding on ME) for copulations in field cages. Without ME, males held without protein for 3 or 7 days obtained significantly fewer matings than control males. With ME, however, males held for even 7 days without protein achieved higher mating success than control males. In a second set of experiments, mature males were held without protein for 7 days and then given a protein-rich diet for 1, 3, or 7 days before testing and were either given or denied access to ME. Without ME, males were competitively inferior to control males when tested 1 or 3 days after resumption of protein feeding and equivalent to control males only after 7 days of protein feeding. With ME, however, males obtained significantly more matings than control males when tested 3 or 7 days after resumed protein feeding and had similar mating success as control males after 1 day of access to the protein-rich diet. Results show that mating success in this species is condition-dependent, with both nutritional state and ME consumption influencing male mating success. Under the test conditions, feeding on ME counteracted a low quality diet and enhanced male mating success.     

Re-examining the relationship between sexual maturation and age of response to methyl eugenol in males of the oriental fruit fly

Males of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), are strongly attracted to, and feed voraciously on, methyl eugenol (ME) and use metabolites of this chemical to synthesize their sex pheromone. Previously, Wong et al. (1989 ) proposed that B .  dorsalis males were attracted to ME even before attaining sexual maturity. However, their interpretation is possibly confounded by the fact that, in monitoring age-related mating readiness, males were presented with equal-aged females. As a result, if females mature more slowly than males, the age of male sexual maturation may have been overestimated, which may have accounted for the discrepancy observed between male age of ME responsiveness and mating activity. Here, we re-examined the relationship between male age and mating readiness by comparing male mating activity when presented with same-aged females vs. sexually mature females. In addition, we measured the age-dependent response of B .  dorsalis males to ME by recording (i) capture in ME-baited traps, and (ii) feeding duration on ME-containing paper discs. Our data support the conclusion of Wong et al. (1989 ) that B .  dorsalis males show attraction to, and feed on, ME before attaining sexual maturity, but suggest that a marked difference in ME response and mating activity exists over a shorter age interval than indicated by Wong et al. (1989 ). Early attraction to, but not ingestion of, ME was related to accelerated sexual maturation. Unexpectedly, ingestion of ME by sexually immature males did not boost their mating success in trials conducted 10 days after feeding on the lure.     

Free radical generation during the activation of hemolymph prepared from the homopteran Dactylopius coccus

Superoxide anion (O(-) (2)) and nitric oxide (NO) generation in Dactylopius coccus hemolymph obtained by perfusion and activated with zymosan was studied. Activated hemolymph reduced 3-[4,5 dimethylthiazolil-2]-2,5-diphenyl tetrazolium bromide. This reduction was prevented by superoxide dismutase (SOD) indicating O(-) (2) generation. This activity was dependent on temperature, and hemolymph incubated at 75 degrees C lost its activity. Chromatocytes incubated with zymosan released their content and produced O(-) (2). Activated hemolymph also produced NO and this activity was prevented in the presence of NG-nitro-L-arginine methyl ester, suggesting that nitric oxide synthase (NOS) might be present in D. coccus hemolymph. The probable source of O(-) (2) in the D. coccus hemolymph is the anthraquinone oxidation, since commercial carminic dye produced O(-) (2) during its oxidation by Agaricus bisporus tyrosinase. Gram+ Micrococcus luteus exposed to activated hemolymph were killed in vitro, and addition of NG-nitro-L-arginine methyl ester and D-Mannitol (a hydroxyl radical scavenger) prevented their killing. The cytotoxic effect produced by the activated hemolymph was not observed with the Gram- bacteria Serratia marcescens. These results suggest that D. coccus activated hemolymph generates reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that may limit M. luteus growth.     

Variability in essential oil composition of Turkish basils (Ocimum basilicum L.)

Sweet basil (Ocimum basilicum L.), one of the most popular aromatic plants, shows great variation in both morphology and essential oil components. In this study, the composition of 18 Turkish basil essential oils was investigated by GC and GC–MS. Variation of essential oils in the landraces was subjected to cluster analysis, and seven different chemotypes were identified. They were (1) linalool, (2) methyl cinnamate, (3) methyl cinnamate/linalool, (4) methyl eugenol, (5) citral, (6) methyl chavicol (estragol), and (7) methyl chavicol/citral. Methyl chavicol with high citral contents (methyl chavicol/citral) can be considered as a “new chemotype” in the Turkish basils. Because methyl eugenol and methyl chavicol have structural resemblance to carcinogenic phenylpropanoids, chemotypes having high linalool, methyl cinnamate or citral contents and a mixture of these is suitable to cultivate for use in industry.     

桔小实蝇雄成虫联系性嗅觉学习

【目的】为了探究桔小实蝇 Bactrocera dorsalis (Hendel)雄成虫的嗅觉学习能力。【方法】本研究采用经典性嗅觉条件反射训练法（classical olfactory conditioning）在室内对固定的羽化后14-17日龄的桔小实蝇雄成虫进行气味与食物的联合学习训练, 即薄荷精油和10%蔗糖溶液联合的奖赏性训练（appetitive conditioning）以及甲基丁香酚(methyl eugenol, ME)和饱和盐溶液联合的惩罚性训练（aversive conditioning）,并以伸喙反射行为（proboscis extension reflex, PER）作为学习与否的判定标准。【结果】经过奖赏性训练后,桔小实蝇雄成虫对薄荷精油的伸喙反射率可从0%增加至68%;而经过惩罚性训练后,桔小实蝇对甲基丁香酚的伸喙反射率可从100%降低至36.54%,且这种伸喙反射率的变化是通过气味条件刺激（conditioned stimulus）和食物非条件刺激（unconditioned stimulus）的对称性联合而产生的。【结论】结果表明,桔小实蝇雄性成虫具有较强的联系性嗅觉学习能力,并且两种刺激的联合是形成学习记忆的必要条件。     

SOURCES OF LARVAL SALIVARY GLAND SECRETION IN THE DIPTERAN CHIRONOMUS TENTANS

The soluble proteins in the hemolymph, the salivary gland, and the salivary secretion of fourth instar Chironomus tentans were examined by disc electrophoresis in acrylamide gels. Of the 11 protein fractions detected in buffered saline extracts of the gland, 10 are present also in the hemolymph. Amino acid isotope incorporation experiments indicate that the protein fractions shared by the salivary gland and the hemolymph are not synthesized in the gland but are synthesized in other larval tissues. Immunochemical studies show that most of these proteins eventually are secreted from the gland. The salivary gland in vivo and in vitro is active in de novo protein synthesis. The protein synthesized tends to form large molecular weight aggregates. As demonstrated by radioautography, at least 80% of this protein is secreted from the 30 large cells forming most of the gland. The proteins synthesized in the salivary gland cannot be detected in the hemolymph. The results of this investigation are consistent with a mechanism of secretion formation involving both de novo synthesis of some secretion proteins and the selective uptake, transport, and secretion of hemal proteins by the salivary gland.