Effects of soybean trypsin inhibitor on hypopharyngeal gland protein content, total midgut protease activity and survival of the honey bee (Apis mellifera L.)

Insecticidal properties of protease inhibitors have been established in transgenic plants. In the wake of continuous research and rapid development of protease inhibitors it is important to assess possible effects on beneficial insects like the honey bee (Apis mellifera L.). In this study, newly emerged caged bees were fed pollen diets containing three different concentrations (0.1%, 0.5% and 1% w:w) of soybean trypsin inhibitor (SBTI). Hypopharyngeal gland protein content, total midgut proteolytic enzyme activity of these bees, and survival were measured. Bees fed 1% SBTI had significantly reduced hypopharyngeal gland protein content and midgut proteolytic enzyme activity. There were no significant differences between control, 0.1% and 0.5% SBTI treatments. Bees fed a diet containing 1% SBTI had the lowest survival, followed by 0.5% and 0.1%, over a 30-day period. We concluded that nurse bees fed a pollen diet containing at least 1% SBTI would be poor producers of larval food, potentially threatening colony growth and maintenance.     

Effects of pollen dilution on infection of Nosema ceranae in honey bees

Multiple stressors are currently threatening honey bee health, including pests and pathogens. Among honey bee pathogens, Nosema ceranae is a microsporidian found parasitizing the western honey bee (Apis mellifera) relatively recently. Honey bee colonies are fed pollen or protein substitute during pollen dearth to boost colony growth and immunity against pests and pathogens. Here we hypothesize that N. ceranae intensity and prevalence will be low in bees receiving high pollen diets, and that honey bees on high pollen diets will have higher survival and/or increased longevity. To test this hypothesis we examined the effects of different quantities of pollen on (a) the intensity and prevalence of N. ceranae and (b) longevity and nutritional physiology of bees inoculated with N. ceranae. Significantly higher spore intensities were observed in treatments that received higher pollen quantities (1:0 and 1:1 pollen:cellulose) when compared to treatments that received relatively lower pollen quantities. There were no significant differences in N. ceranae prevalence among different pollen diet treatments. Interestingly, the bees in higher pollen quantity treatments also had significantly higher survival despite higher intensities of N. ceranae. Significantly higher hypopharyngeal gland protein was observed in the control (no Nosema infection, and receiving a diet of 1:0 pollen:cellulose), followed by 1:0 pollen:cellulose treatment that was inoculated with N. ceranae. Here we demonstrate that diet with higher pollen quantity increases N. ceranae intensity, but also enhances the survival or longevity of honey bees. The information from this study could potentially help beekeepers formulate appropriate protein feeding regimens for their colonies to mitigate N. ceranae problems.     

Effect of primer pheromones and pollen diet on the food producing glands of worker honey bees (Apis mellifera L.)

Cooperative brood care is highly developed in the honey bee such that workers called nurses use their hypopharyngeal and mandibular glands to biosynthesize proteinaceous secretions that are progressively provisioned to larvae. The role that honey bee primer pheromones play in the functional physiology of food producing glands was examined. The combined and separate effects of queen mandibular pheromone (QMP) and brood pheromone (BP) on amount of protein extractable from hypopharyngeal and mandibular glands of workers reared for 12 days with and without pollen diets was measured. In rearing environments with a pollen diet, BP, and QMP + BP pheromone treatments significantly increased extractable protein from both glands. Bees reared with QMP + pollen had amounts of protein extractable from both glands that were not significantly different from control bees (no pheromones, no pollen). Pollen in the diet alone significantly increased amounts of protein extractable from glands versus control. In rearing environments without pollen, QMP + BP had a synergizing effect on amount of protein in both glands. The QMP + BP treatment was the only rearing environment without a pollen diet where protein amounts were significantly greater than the control. The synergizing effect of QMP + BP on extractable mandibular and hypopharyngeal gland protein suggests a highly derived role for the combined effect of these two primer pheromones on honey bee cooperative brood care. Mandibular gland area was significantly and positively correlated with extractable protein. Amounts of extractable protein from both glands declined significantly with age of workers in all treatments. However, treatment significantly affected rate of decline. The adaptive significance of gland protein amounts in response to pheromones and pollen diet are discussed.     

Effects of time, temperature, and honey on Nosema apis (Microsporidia: Nosematidae), a parasite of the honeybee, Apis mellifera (Hymenoptera: Apidae).

Newly emerged adult bees were fed with Nosema apis spores subjected to various treatments, and their longevity, proportions of bees infected, and spores per bee recorded. Spores lost viability after 1, 3, or 6 months in active manuka or multifloral honey, after 3 days in multifloral honey, and after 21 days in water or sugar syrup at 33 degrees C. Air-dried spores lost viability after 3 or 5 days at 40 degrees, 45 degrees, or 49 degrees C. Increasing numbers of bees became infected with increasing doses of spores, regardless of their subsequent food (active manuka honey, thyme honey, or sugar syrup). Final spore loads were similar among bees receiving the same food, regardless of dose. Bees fed with either honey had lighter infections than those fed with syrup, but this may have been due to reductions in their longevity. Bees fed with manuka honey were significantly shorter lived, whether infected or not.     

The provision of supplementary food to hive bees

Sucrose syrup is less satisfactory than honey as a winter and spring food for bees: a mixture of approximately equal parts of honey and syrup is of almost the same value as honey alone.
Feeding confined to early autumn induced the strongest spring development of colonies; syrup feeding in the spring may retard colony development, and food supplied at this time is apparently wasted. Feeding with syrup and pollen is advantageous only when a colony is lacking in stores of carbohydrate and protein.
A total of four British Standard brood combs full of pollen provides sufficient protein for a colony on B.S. equipment from autumn until April: the best results were obtained by providing colonies on eleven B.S. combs, in early autumn, with 35-40 lb. of honey, or honey and concentrated sucrose syrup, and four brood combs full of pollen.     

Ecologically appropriate xenobiotics induce cytochrome P450s in Apis mellifera

Background

Honey bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored.

Methodology/Principal Findings

We used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey.

Conclusions/Significance

These results suggest that regulation of honey bee P450s is tuned to chemicals occurring naturally in the hive environment and that, in terms of toxicological capacity, a diet of sugar is not equivalent to a diet of honey.     

In vivo Responses of Honey Bee Midgut Proteases to Two Protease Inhibitors from Potato

Potato protease inhibitors, POT-1 and POT-2, were fed to newly emerged adult honey bees in cages at different doses in either sugar syrup (0.2 or 0.01% w:v) or pollen food (1 or 0.2% w:w). In vivo activities of three digestive endopeptidases (trypsin, chymotrypsin and elastase) and one exopeptidase (leucine aminopeptidase; LAP) were measured after 3 or 8days' exposure of bees to inhibitor. Enzyme activities were significantly lower at day 8 than at day 3, except for elastase, which did not change. POT-2 significantly reduced the activity of all endopeptidases at both timepoints, regardless of the dose level or the medium in which the inhibitor was administered. POT-1 acted in a similar manner, except that 0.01% POT-1 in syrup had no effect on bees. There was no consistent trend in changes in LAP activity. Bees fed either inhibitor at 1% in pollen or at 0.2% in syrup had significantly reduced lifespans, with the effect of the pollen treatment being greater than the syrup treatment. The survival of bees fed POT-1 or POT-2 at 0.2% in pollen or 0.01% in syrup did not differ from the controls.     

Post‐ingestive effect of plant phenolics on the feeding behaviour of the honeybee Apis cerana

As the staple food of honey bees, honey is rich in plant phenolics derived from pollen, nectar and resin. Most studies concentrate on the temporary response of bees' peripheral chemoreceptors to these chemicals, and the post‐ingestive effects of plant phenolics are largely ignored. In the present study, a series of feeding experiments are conducted to test whether plant phenolics modulate the response thresholds and rhythmic behaviour of the honeybee Apis cerana (Ruttner). The results of the study demonstrate that bees fed with syrup containing high concentrations of phenlics reduce their response thresholds greatly, and shift their feeding rhythms significantly. Because the forager response thresholds determine their foraging choice, and their rhythmic behaviour is required for timing visits to flowers, enhanced plant phenolics as a result of global environment change may change the bees' pollination service in our changing world.     

Nurse bees support the physiological development of young bees (Apis mellifera L.)

Newly emerged worker honeybees (focal bees) were caged individually for 8 days either isolated or together with one companion bee of known age (2–30 days) taken from a colony. The companion bee was replaced every 2nd day. After 8 days, various parameters were investigated in the focal bees as indicators of the level of development. Focal bees which had been caged with 6-day-old companion bees were better developed than isolated focal bees, newly emerged bees, or focal bees caged with almost all other ages of companion bees. They had hypopharyngeal glands that were larger and contained more protein, their thoraces had a higher protein content, and they had a higher rate of proteolytic activity in the midgut. Although the focal bees were supplied with pollen as well as honey, they consumed only small amounts of pollen. We attribute their better development to their having been fed worker jelly by the accompanying companion bees. The 6-day-old companion bees consumed high quantities of pollen and spent more time (18.7 ± 11.85 s/h) feeding focal bees than 12-day-old bees (6.5 ± 4.09 s/h) or foragers (no feeding of focal bees). The results show that even under such artificial conditions, the exchange of food (trophallaxis) promotes the development of young honeybee workers. Accepted: 26 February 1999     

Behavior and molecular physiology of nurses of worker and queen larvae in honey bees (Apis mellifera)

In a honey bee colony, worker bees rear a new queen by providing her with a larger cell in which to develop and a large amount of richer food (royal jelly). Royal jelly and worker jelly (fed to developing worker larvae) differ in terms of sugar, vitamin, protein and nucleotide composition. Here we examined whether workers attending queen and worker larvae are separate specialized sub-castes of the nurse bees. We collected nurse bees attending queen larvae (AQL) and worker larvae (AWL) and compared gene expression profiles of hypopharyngeal gland tissues, using Solexa/Illumina digital gene expression tag profiling (DGE). Significant differences in gene expression were found that included a disproportionate number of genes involved in glandular secretion and royal jelly synthesis. However behavioral observations showed that these were not two entirely distinct populations. Nurse workers were observed attending both worker larvae and queen larvae, and there was no evidence of a specialized group of workers that preferentially or exclusively attended developing queens. Nevertheless, AQL attended larvae more frequently compared to AWL, suggesting that nurses sampled attending queen larvae may have been the most active nurses. This study serves as another example of the relationship between differences in gene expression and behavioral specialisation in honey bees.     

Nutritional effects of supplementary diets on brood development,biological activities and honey production of Apis mellifera L

The present research work was conducted to assess the impact of nutrient-enriched diet on the physiological activities and subsequently honey yield. Eighteen colonies of Apis mellifera L. were selected from Dera Ismail Khan region, KPK, Pakistan, during the winter and summer seasons, 2019–2020. Five pollen supplement diets were prepared and provided to screen out the palatable one to be fed as pollen alternative nutrition to bee bread. Results of diet consumption regarding mean data for consumption rate displayed that soybean flour enriched artificial diet was maximally consumed (74.34 g) by honey bees per week. Minimum consumption was observed for grinded groundnut enriched diet (64.62 g) which was relatively lesser than the other tested artificial diets. Results of area of worker brood disclosed that soybean flour fortified diet (1489.27 cm²/colony) statistically noteworthy than the other artificial diets whereas control (463.51 cm²/colony) was least effective. Highest bee strength (10.00 bee frames/colony) was noted in the bee colonies fed with soybean flour fortified diet, date paste (8.0 bee frames/colony) was the next effective one, among the tested pollen replacement diets whereas relatively least (5 bee frames/colony) was noticed in case of grinded groundnut. Highest body weight (12.41 g) of neonate bees was noted in case of soybean enriched diet while lowermost (5.31 g) was noted in control bees. Results of wax cell built up and foraging efficiency were also superior in artificial diets than respective control bees. Hence, artificial diets especially soybean-enriched pollen alternative diet can boost up the physiology of honey bee leading to increased honey yield and profit.     

Lipid-enhanced pollen and lipid-reduced flour diets and their effect on the longevity of honey bees (Apis mellifera L.)

Abstract  As eucalypt pollens contain low concentrations of lipid, enhancing pollen diets with fatty acids was hypothesised to improve honey bee longevity. Different concentrations of linoleic and oleic acid added to eucalypt pollen were trialled in small cages containing approximately 1400 bees each. Bees fed diets of redgum ( Corymbia calophylla (Lindl.) Hill & Johnson, formerly Eucalyptus calophylla ) pollen had the lowest mortality of 22 diets tested for 6 weeks and had life spans (50%) greater than 42 days. Linoleic acid mixed with a redgum diet in concentrations >6% corresponded to life spans of 24–25 days. Bee longevity appeared to be more sensitive to oleic acid as life spans decreased to 15–21 days when diets had concentrations >2%. The life spans of bees fed soya bean flour were 26 days on low (0.6% lipid) fat, 19 days on defatted and 20 days on full-fat diets. Bees fed lupin flour had a life span of 23 days. Adding redgum pollen to lupin flour caused increased mortality, but addition of pollen to soya bean flour was beneficial. Thus, beekeepers who choose to utilise soya bean or lupin flours as protein substitutes to pollen will have bees with reduced longevity. Bees fed redgum pollen that had been dried, crushed, irradiated and hermetically stored in a cool room for several years had similar longevity to bees fed fresh-collected and frozen redgum pollen.     

取食蜂蜜对棉铃虫齿唇姬蜂体内主要代谢物质的影响

为揭示食物在齿唇姬蜂Campoletis chlorideae Uchida生命中所起的作用,测定分析饱食蜂蜜和只取食清水的齿唇姬蜂体内糖类、脂肪和蛋白质随日龄增加的变化趋势。结果显示,取食蜂蜜对齿唇姬蜂体内主要代谢物质影响显著。10%蜂蜜水和清水饲养的齿唇姬蜂的雌蜂和雄蜂在刚羽化时体内都含有较高水平的糖原、总糖、脂肪和蛋白质。但随着日龄的增加,用清水饲养的雌、雄蜂体内的糖原和总糖含量迅速下降并达到最低水平;而用蜂蜜水饲养的雌、雄蜂随日龄的增加体内的糖原含量没有显著变化,并且与清水饲养的雌、雄蜂相比,其体内总糖含量反而有所上升。用蜂蜜水饲养的齿唇姬蜂的寿命显著长于用清水饲养的齿唇姬蜂。由于蜂蜜中含有丰富的果糖,所以冷蒽酮反应测定显示,用蜂蜜水饲养的齿唇姬蜂体内果糖含量较高,但却不能在清水饲养的齿唇姬蜂体内测出。2种食物处理的齿唇姬蜂在羽化后体内脂肪含量均有下降;并且随日龄的增加,清水饲养的齿唇姬蜂体内脂肪含量的下降速度明显快于蜂蜜水饲养的齿唇姬蜂。取食蜂蜜水的齿唇姬蜂体内蛋白质含量随日龄无显著变化;而用清水饲养的齿唇姬蜂在羽化后体内蛋白质含量随日龄的增长迅速下降并达到最低水平。     

不同饲料对小峰熊蜂工蜂群发育的影响

糖和花粉对熊蜂的生长发育和繁殖起重要作用。本研究测定和分析了2种糖源（白砂糖、蜂蜜）和5种花粉（杏花粉、油菜花粉、向日葵花粉、玉米花粉、茶花粉）的饲料组合对小峰熊蜂Bombus hypocrita的无王工蜂群寿命、产卵前期时间、产卵量、幼虫拖出数、卵杯数、雄蜂出房时间和子代雄蜂数量的影响。结果表明： 只饲喂糖类食物时, 工蜂寿命显著短于有花粉的饲料组, 而且工蜂不产卵。含有花粉的各饲料组之间工蜂寿命存在显著差异, 饲喂玉米花粉的工蜂寿命小于其他4种花粉组。在工蜂产卵前期时间和卵杯数量方面, 10种含有花粉的饲料组之间差异不显著; 但在产卵量、幼虫拖出数、雄蜂出房时间和子代雄蜂数量等方面, 各饲料组之间差异较大。白砂糖和杏花粉组的产卵量最高; 杏花粉组和茶花粉组的幼虫拖出数量显著低于其他花粉组; 油菜花粉组和杏花粉组的雄蜂出房时间显著短于其他花粉组; 杏花粉组的子代雄蜂数量显著高于其他饲料组。结果显示不同饲料组合对小峰熊蜂工蜂群的发育影响很大。因此建议在熊蜂的人工繁育过程中, 在不同的发育阶段应给予不同的饲料配方。     

Field and semifield evaluation of impacts of transgenic canola pollen on survival and development of worker honey bees

A 2-yr field trial (2001 and 2002) and 1-yr semifield trial (2002) were conducted to evaluate the effect of transgenic herbicide (glyphosate) -tolerant canola Brassica napus L. pollen on larval and adult honey bee, Apis mellifera L., workers. In the field trial, colonies of honey bees were moved to transgenic or nontransgenic canola fields (each at least 40 hectares) during bloom and then sampled for larval survival and adult recovery, pupal weight, and hemolymph protein concentrations. No differences in larval survival, adult recovery, and pupal weight were detected between colonies placed in nontransgenic canola fields and those in transgenic canola fields. Colonies placed in the transgenic canola fields in the 2002 field experiment showed significantly higher hemolymph protein in newly emerged bees compared with those placed in nontransgenic canola field; however, this difference was not detected in the 2001 field experiment. In the semifield trial, bee larvae were artificially fed with bee-collected transgenic and nontransgenic canola pollen and returned to their original colonies. Larval survival, pupal survival, pupal weight, and hemolymph protein concentration of newly emerged adults were measured. There were no significant differences in any of the parameters measured between larvae that were fed transgenic canola pollen and those fed nontransgenic corn pollen. Results from this study suggest that transgenic canola pollen does not have adverse effects on honey bee development and that the use of transgenic canola dose not pose any threat to honey bees.     

Influence of age and caging upon protein metabolism,hypopharyngeal glands and trophallactic behavior in the honey bee (Apis mellifera L.)

Summary Different-aged honey bees were either kept in a cage together with young sisters for eight days or lived in their colony. Following an injection of¹⁴C-phenylalanine (Phe) we measured incorporation of¹⁴C-Phe into head protein and total protein, as well as the size of the hypopharyngeal glands. While confined in a cage for four hours, injected bees (from colony or cage) dispensed the¹⁴C-labelled protein-rich products of their hypopharyngeal glands to recipients. Eight-day-old colony bees had well developed hypopharyngeal glands, whereas at the age of sixteen days the glands had already decreased in size. Young caged bees had smaller hypopharyngeal glands. Colony bees had higher incorporation rates into total protein and head protein than bees living in a cage. Bees of different age classes, irrespective of caging, fed the same number of recipients; but the amount of¹⁴C-labelled protein-rich jelly distributed by caged bees was significantly smaller than that distributed by colony bees. Our results indicate that trophallaxis between young donor workers and newly emerged recipient worker bees is not the key factor for regular development and activity of the hypopharyngeal glands.Dedicated to Achim Lass's daughter Katrin.     

感染囊幼病的工蜂咽下腺细胞的超微结构的变化

前言 中华蜜蜂囊幼病是我国养蜂业的一种重要病毒病,发病率很高,有的地区造成大量的幼虫死亡,给我国养蜂生产带来一定的危害,为了对防治病害提供科学的依据,我们首先对该病病原进行了分离、提纯及电子显微镜的研究等工作。发现该病毒对幼虫和成蜂,尤其对工蜂体内各个器官都有程度不同的影响。在幼虫发病期症状特别明显,病幼虫身体松软多水,其表皮容易破裂,当悬挂幼虫时其幼虫末端积聚有透明的液滴,在巢房内幼虫头部尖并变成黑色,头部稍微向上抬起呈船形。死后的幼虫干涸变为褐色的外壳留于巢房内,当感染的幼虫全部封盖后,巢房盖的中央有一小孔,这些异常的变化都是囊幼病的典型症状。幼虫症状虽很明显,但工蜂感染病毒后在外部形态上却没有明显     

Consumption rate of some proteinic diets affecting hypopharyngeal glands development in honeybee workers

The experiment was carried out under laboratory condition to study the consumption of some proteinic diets and their effect on hypopharyngeal glands (HPG) development during nursing period. The results showed that the bee bread and the pollen loads mixture with sugar (1:1) were more consumed by honeybee workers followed by Nectapol® and Yeast-Gluten mixture. The lowest consumption amount was recorded with traditional substitute.Clear differences were found in HPG development under feeding with different diets. The maximum development degree was observed when fed with bee bread followed by pollen loads and mixture from Yeast, Gluten and sugar (1:1:2). The acinal surface of HPG showed clear difference under feeding with difference diets. The largest area was recorded when honeybee workers fed on bee bread followed by Yeast-Gluten-sugar mixture (diet,4) and pollen loads(diet,2).     

Regulation of hypopharyngeal gland activity and oogenesis in honey bee (Apis mellifera) workers

In the honey bee, vitellogenin has several functions in addition to egg provisioning. Among others, it serves as a precursor to brood food proteins secreted by the hypopharyngeal glands of worker bees. In queenless workers with developing gonads, oogenesis and development of the hypopharyngeal glands are correlated. Here we describe two experiments that explored whether this relationship also exists in non-reproductive workers, and investigated a possible role of ecdysteroid hormones in the regulation of vitellogenin uptake. In the first experiment, the correlation between oocyte length and hypopharyngeal gland development was measured in workers before and after de-queening. In the second experiment, we induced middle-aged bees with resting glands to suddenly initiate brood care behaviour, and measured haemolymph ecdysteroid and vitellogenin titres. A strong positive relationship existed between morphometrical parameters of hypopharyngeal glands and ovaries in both queenless and queenright (functionally sterile) workers. No response of ecdysteroid titres to the addition of brood was detected in experiment 2, but high concentrations were measured in a small group of bees characterised by the possession of oocytes on the brink of yolk incorporation. We conclude that hypopharyngeal glands may belong to a previously described group of reproduction-related traits that are pleiotropically regulated in workers. A possible role for ecdysteroids in honey bee reproduction is discussed.     

Physiological correlates of division of labor among similarly aged honey bees

Hormone analyses and exocrine gland measurements were made to probe for physiological correlates of division of labor among similarly aged adult worker honey bees (Apis mellifera L.). Middle-age bees (ca. 2 weeks old) performing different tasks showed significant differences in both juvenile hormone (JH) biosynthesis rates and hemolymph titers; guards and undertakers had high JH, and wax producers and food storers, low JH. Guards and undertakers had similar hormone levels to foragers, even though they were 10 days younger than foragers. No differences in JH were detected among young bees (1-week-old queen attendants and nurses) or older bees (3–4 week-old pollen foragers, non-pollen foragers, and soldiers). Hypopharyngeal gland size was inversely correlated with worker age and rate of JH biosynthesis, but soldiers had significantly larger hypopharyngeal glands than did foragers, despite their similar age and JH level. Results from soldiers indicate that exocrine gland development is not always linked with age-related behavior and endocrine development; they also support the recent claim that soldiers constitute a group of older bees that are distinct from foragers. Hormonal analyses indicate that the current model of JH's role in honey bee division of labor needs to be expanded because high levels of JH are associated with several other tasks besides foraging. JH may be involved in the regulation of division of labor among similarly aged workers in addition to its role in age-related division of labor.Abbreviations JH Juvenile hormone - RIA radioimmunoassay - CA corpora allata - HPLC high performance liquid chromatography - TLC thin layer chromatography