Abstract

Social organization in highly eusocial bees relies upon two important processes: caste differentiation in female larvae, and age polyethism in adult workers. Juvenile Hormone (JH) is a key regulator of both processes. Here we investigated the expression of two genes involved in JH metabolism - mfe (biosynthesis) and jhe (degradation) - in the context of social organization in the stingless bee Melipona interrupta. We found evidence that the expression of mfe and jhe genes is related to changes in JH levels during late larval development, where caste determination occurs. Also, both mfe and jhe were upregulated when workers engage in intranidal tasks, but only jhe expression was downregulated at the transition from nursing to foraging activities. This relation is different than expected, considering recent reports of lower JH levels in foragers than nurses in the closely related species Melipona scutellaris. Our findings suggest that highly eusocial bees have different mechanisms to regulate JH and, thus, to maintain their level of social organization.

Keywords:
Juvenile hormone; eusocial bees; division of labor; caste determination; age polyethism

Introduction

Juvenile Hormone (JH) is an isoprenoid compound that plays a remarkable role in the development (including metamorphosis), reproduction and regulation of distinct morphologies and behaviors of insect species (Hartfelder, 2000Hartfelder K (2000) Insect juvenile hormone: From “status quo” to high society. Braz J Med Biol Res 33:157-177.; Truman and Riddiford, 2002Truman JW and Riddiford LM (2002) Endocrine insights into the evolution of metamorphosis in insects. Annu Rev Entomol 47:467-500.). JH is synthetized in the corpora allata (CA), which are endocrine glands located in the posterior regions of the insect head, and transported through the hemolymph to its site of action in target cells (Tobe and Stay, 1985Tobe SS and Stay B (1985) Structure and regulation of the corpus allatum. Adv In Insect Phys 18:305-432.; Gilbert et al., 2000Gilbert LI, Granger NA and Roe RM (2000) The juvenile hormones: Historical facts and speculations on future research directions. Insect Biochem Mol Biol 30:617-644.). The presence or absence of JH at precise time points during the life cycle of insects is essential for its function. JH levels are ultimately regulated by two biochemical processes: JH biosynthesis and JH degradation (Gilbert et al., 2000Gilbert LI, Granger NA and Roe RM (2000) The juvenile hormones: Historical facts and speculations on future research directions. Insect Biochem Mol Biol 30:617-644.).

JH production has been considered the main mechanism for controlling JH titers (Gilbert et al., 2000Gilbert LI, Granger NA and Roe RM (2000) The juvenile hormones: Historical facts and speculations on future research directions. Insect Biochem Mol Biol 30:617-644.). In CA, JH is synthetized via the mevalonate metabolic pathway, which involves 13 enzymatic steps. The final and critical step of JH biosynthesis is catalyzed by the enzyme Methyl Farnesoate Epoxidase (MFE) (Bellés et al., 2005Bellés X, Martín D and Piulachs M-D (2005) The mevalonate pathway and the synthesis of juvenile hormone in insects. Annu Rev Entomol 50:181-199.). JH degradation, on the other hand, is catalyzed primarily by one of two enzymes: JH Esterase (JHE) or JH Epoxide Hydrolase (JHEH) (Kort and Granger, 1981Kort CAD and Granger NA (1981) Regulation of the juvenile hormone titer. Annu Rev Entomol 26:1-28. ; Gilbert et al., 2000Gilbert LI, Granger NA and Roe RM (2000) The juvenile hormones: Historical facts and speculations on future research directions. Insect Biochem Mol Biol 30:617-644.). JHE is released from fat body to act in hemolymph and other tissues, and it is considered the primary pathway of JH inactivation, while JHEH activity is secondary and exclusively tissue-bound (Tobe and Stay, 1985Tobe SS and Stay B (1985) Structure and regulation of the corpus allatum. Adv In Insect Phys 18:305-432.).

For its wide function in the life cycle of insects and responsiveness to environmental stimuli, JH acquired pleiotropic functions within species, such as wing polyphenism in aphids, horn length in beetles, flight activity in moths, and regulation of social organization in many Hymenoptera species (Hartfelder, 2000Hartfelder K (2000) Insect juvenile hormone: From “status quo” to high society. Braz J Med Biol Res 33:157-177.). In highly eusocial bees (honey bees and stingless bees), social organization within colonies is based mainly on two processes: caste differentiation, in which females are morphologically specialized into reproductive or non-reproductive tasks (related to brood care and colony maintenance), and age polyethism, by which adult workers perform different tasks during their lives according to their ages (Robinson and Vargo, 1997Robinson GE and Vargo EL (1997) Juvenile hormone in adult eusocial Hymenoptera: Gonadotropin and behavioral pacemaker. Arch Insect Biochem Physiol 35:559-583.).

In the honey bee Apis mellifera, caste differentiation is triggered by nutritional signals. Queen-destined larvae are fed with royal jelly during their entire larval development, while worker-destined larvae stop being fed with royal jelly in the late larval stages (Hartfelder and Emlen, 2012Hartfelder K and Emlen DJ (2012) Endocrine control of insect polyphenism. In: Gilbert LI (ed) Insect Endocrinology. Academic Press, London, pp 464-522.). These distinct nutritional signals generate changes in JH patterns. When JH titers are above a certain threshold, this leads to queen development (Hartfelder and Emlen, 2012Hartfelder K and Emlen DJ (2012) Endocrine control of insect polyphenism. In: Gilbert LI (ed) Insect Endocrinology. Academic Press, London, pp 464-522.). Age polyethism in honey bees, in turn, is mainly characterized by young workers performing tasks within the hive, such as brood care and nest maintenance (nursing); while older workers perform activities outside the hive, such as food collection and nest defense (foraging) (Robinson and Vargo, 1997Robinson GE and Vargo EL (1997) Juvenile hormone in adult eusocial Hymenoptera: Gonadotropin and behavioral pacemaker. Arch Insect Biochem Physiol 35:559-583.). In A. mellifera, low hemolymph JH titers are associated with nursing tasks, while high JH titers are associated with foraging activities (Rutz et al., 1976Rutz W, Wille LGH and Scher ML (1976) The function of juvenile hormone in adult worker honeybees, Apis mellifera. J Insect Physiol 22:1485-1491.; Huang et al., 1991Huang ZHY, Robinson GE, Tobe SS, Yagi KJ, Strambi C, Strambi A and Stay B (1991) Hormonal regulation of behavioural development in the honeybee is based on changes in the rate of juvenile hormone biosynthesis. J Insect Physiol 37:733-741.).

In most stingless bees, caste determination is also triggered by differential feeding, but only by quantitative, not qualitative differences. Females that receive larger quantities of food develop into queens (Camargo et al., 1976Camargo CA, Almeida MG de, Parra MGN and Kerr WE (1976) Genetics of sex determination in bees. IX. Frequencies of queens and workers from larvae under controlled conditions (Hymenoptera: Apoidea). J Kansas Entomol Soc 49:120-125.). In Melipona bees, however, caste is not primarily determined by differences in food quantity or quality (Kerr, 1950Kerr WE (1950) Genetic determination of castes in the genus Melipona. Genetics 35:143-152.; Camargo et al., 1976Camargo CA, Almeida MG de, Parra MGN and Kerr WE (1976) Genetics of sex determination in bees. IX. Frequencies of queens and workers from larvae under controlled conditions (Hymenoptera: Apoidea). J Kansas Entomol Soc 49:120-125.; Maciel-Silva and Kerr, 1991Maciel-Silva VL and Kerr WE (1991) Sex determination in bees. XXVII. Castes obtained from larvae fed homogenized food in Melipona compressipes (Hymenoptera, Apidae). Apidologie 22:15-19.). A genetic model with two-loci/two-alleles is the most accepted hypothesis so far to explain caste determination (Jarau et al., 2010Jarau S, van Veen JW, Twele R, Reichle C, Gonzales EH, Aguilar I, Francke W, Ayasse M, Veen JW Van, Twele R et al. (2010) Workers make the queens in Melipona bees: Identification of geraniol as a caste determining compound from labial glands of nurse bees. J Chem Ecol 36:565-569.). According to it, females that are homozygous at one or two loci turn into workers, while females that are double heterozygous (and well fed), develop into queens (Kerr et al., 1966Kerr WE, Stort AC and Montenegro MJ (1966) Importância de alguns fatores ambientais na determinação das castas do gênero Melipona. An Acad Bras Cienc 38:149-168.; Kerr et al., 1975Kerr WE, Akahira Y and Camargo CA (1975) Sex determination in bees. IV. Genetic control of juvenile hormone production in Melipona quadrifasciata (Apidae). Genetics 81:749-756.). Regardless of the initial input, high JH titers also lead to the development of queens in stingless bee species (Campos, 1978Campos LAO (1978) Sex determination in bees. VI. Effect of a juvenile hormone analogue in males and females of Melipona quadrifasciata (Apidae). J Kansas Entomol Soc 51:228-234.; Bonetti, 1984Bonetti AM (1984) Efeitos do hormônio juvenil no desenvolvimento ovariano de Melipona quadrifasciata. Rev Bras Biol 44:509-516.). Age polyethism in stingless bees, on the other hand, has a very similar pattern to the one observed in honey bees, with the transition from nest activities to foraging as workers gets older (Kerr and Santos-Neto, 1956Kerr WE and Santos-Neto GR (1956) Contribuição para o conhecimento da bionomia dos Meliponini 5. Divisão de trabalho entre as operárias de Melipona quadrifasciata Lep. Insectes Soc 3:423-430.). The endocrine basis of age polyethism was investigated in adult workers of Melipona scutellaris (Cardoso-Júnior et al., 2017Cardoso-Júnior ACM, Silva RP, Borges NA, Carvalho WJ, Walter SL, Simões ZLP, Bitondi MMG, Vieira CU, Bonetti AM and Hartfelder K (2017) Methyl farnesoate epoxidase (mfe) gene expression and juvenile hormone titers in the life cycle of a highly eusocial stingless bee, Melipona scutellaris. J Insect Physiol 101:185-194.). However, in contrast to honey bees, JH titers were significantly higher in nurses than in foragers.

Considering that JH has a crucial role for caste development and behavioral division of labor in highly eusocial bees, regulation of JH levels should be critical for the control of their social organization. Studies about the expression of mfe and jhe genes were done mainly with honey bees, with evidence that the transcription of these genes might be key regulation points of JH titers in the social organization context (Mackert et al., 2008Mackert A, Nascimento AM, Bitondi MMG, Hartfelder K and Simões ZLP (2008) Identification of a juvenile hormone esterase-like gene in the honey bee, Apis mellifera L. - Expression analysis and functional assays. Comp Biochem Physiol Part B Biochem Mol Biol 150:33-44.; Bomtorin et al., 2014Bomtorin AD, Mackert A, Rosa GCC, Moda LM, Martins JR, Bitondi MMG, Hartfelder K and Simões ZLP (2014) Juvenile hormone biosynthesis gene expression in the corpora allata of honey bee (Apis mellifera L.) female castes. PLoS One 9:e86923. ). Thus, this study aimed to investigate the transcriptional regulation of mfe and jhe genes in critical moments for caste differentiation and age-related division of labor in adult workers, and its importance for social organization in the Amazonian stingless bee Melipona interrupta.

Material and Methods

Bee sampling

Larvae and adult samples of M. interrupta were obtained from the meliponary of the Bee Research Group (GPA) located at the National Institute of Amazonian Research (INPA), Manaus/AM/Brazil (-3.0973067, -59.9847846). Larvae were collected at the second and the third instar, which is the last (and longest) in Melipona bees (n = 30) (Dias et al., 2001Dias JD, Bonetti AM and Kerr WE (2001) Determinação do número de instares larvais em Melipona scutellaris (Hymenoptera: Apidae). Naturalia 26:257-263.). The subdivisions of the third instar were classified based on the food amount/consistency and the presence/absence of feces within the brood cells, in accordance with previous studies with Melipona bees (Amaral et al., 2010Amaral IMR, Moreira Neto JF, Pereira GB, Franco MB, Beletti ME, Kerr WE, Bonetti AM and Ueira-Vieira C (2010) Circulating hemocytes from larvae of Melipona scutellaris (Hymenoptera, Apidae, Meliponini): Cell types and their role in phagocytosis. Micron 41:123-129.; Cardoso-Júnior et al., 2017Cardoso-Júnior ACM, Silva RP, Borges NA, Carvalho WJ, Walter SL, Simões ZLP, Bitondi MMG, Vieira CU, Bonetti AM and Hartfelder K (2017) Methyl farnesoate epoxidase (mfe) gene expression and juvenile hormone titers in the life cycle of a highly eusocial stingless bee, Melipona scutellaris. J Insect Physiol 101:185-194.) (Table 1). Adult workers were sampled at three stages (n = 27). Newly emerged workers were collected after they hatched from brood cells; nurses were captured while constructing or provisioning cells with larval food; and foragers were caught while returning to the nest with nectar or pollen. All individuals were stored at -80°C after sampling.

Identification of mfe and jhe transcripts in M. interrupta

Total RNA was extracted from whole body of each individual with the SV RNA Isolation System kit (Promega), following the manufacturer’s instructions. RNA samples were quantified in the spectrophotometer Nanodrop 2000 (Thermo Fisher Scientific) and their integrity was verified by electrophoresis in agarose gel 1% (w/v). First-strand cDNA synthesis was made from 1 µg of total RNA using the Improm-II™ Reverse Transcription System kit (Promega).

Since there was no genome sequence available for M. interrupta, primers were initially designed to amplify and sequence partially the cDNA of mfe and jhe in this species, based on the coding sequences of orthologous genes from the closely related species M. quadrifasciata (^{Table S1} Table S1 - Primers used in PCR (gene identification) and qRT-PCR (gene expression) for the analysis of mfe and jhe in Melipona interrupta. ). The OrthoDBOrthoDB: Database of Orthologous Groups, http://cegg.unige.ch/orthodb7
http://cegg.unige.ch/orthodb7... : Database of Orthologous Groups program was employed to search for the mfe sequence in the genome of M. quadrifasciata, using mfe sequence of Apis mellifera as query (GenBank accession number: NM_001327966.1). The jhe sequence of M. quadrifasciata was obtained through a search directly in GenBank (accession number: EU099312.1). Sequencing was carried out on an ABI 3130 Genetic Analyzer (Applied Biosystems) with Big Dye^® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), following manufacturer’s instructions. The obtained sequences were compared to the sequences deposited in GenBank database using the Blast toolBasic Local Alignment Search Tool (BLAST), https://blast.ncbi.nlm.nih.gov/Blast.cgi
https://blast.ncbi.nlm.nih.gov/Blast.cgi... .

Expression of mfe and jhe in larvae and adults of M. interrupta

The expression of mfe and jhe was analyzed using the method of quantitative real time PCR (two-step qRT-PCR). Primer sequences for mfe and jhe amplification were specific for M. interrupta (^{Table S1} Table S1 - Primers used in PCR (gene identification) and qRT-PCR (gene expression) for the analysis of mfe and jhe in Melipona interrupta. ). Amplification detection was made with the Platinum^® SYBR^® Green qPCR Super Mix-UDG kit (Invitrogen), using the following reagent concentrations: 0.8X of SYBR^® green master mix, 0.5 µM of ROX reference dye, 0.4 µM of each primer, cDNA (0.25 µL generated from 1 µg of total RNA) and nuclease-free water q.s. 12.5 µL. Each reaction was done three times (technical replicates). Reactions were carried out on a Step One™ Real-Time PCR System (Thermo Fisher Scientific) in the following conditions: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C of 30 s. Melting curves were made at the end of each run to confirm the specificity of PCR products. Amplification efficiency (E) was estimated by using standard curves from a 1:3 dilution series of a representative sample and applying the equation E = 10^[-1/slope]. E values were 98% and 109,2% for mfe and jhe primers pairs, respectively.

Expression levels were measured by the relative quantification method. rpL32 (ribosomal protein L32) and act (actin) were used as reference genes for normalization, which were already experimentally validated as genes with stable expression in all developmental stages in M. interrupta (Brito et al., 2015Brito DV., Silva CGN, Hasselmann M, Viana LS, Astolfi-Filho S and Carvalho-Zilse GA (2015) Molecular characterization of the gene feminizer in the stingless bee Melipona interrupta (Hymenoptera: Apidae) reveals association to sex and caste development. Insect Biochem Mol Biol 66:24-30.). Primers for rpL32 (previously named rp49) and act amplification were taken from literature data (Lourenço et al., 2008Lourenço AP, Mackert A, Cristino AS and Simões ZLP (2008) Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR. Apidologie 39:372-385.; Brito et al., 2015Brito DV., Silva CGN, Hasselmann M, Viana LS, Astolfi-Filho S and Carvalho-Zilse GA (2015) Molecular characterization of the gene feminizer in the stingless bee Melipona interrupta (Hymenoptera: Apidae) reveals association to sex and caste development. Insect Biochem Mol Biol 66:24-30.). Relative gene expression was calculated using the formula proposed by Pfaffl (2001Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:16-21.), which allows the adjustment of differences between amplification efficiencies of target and reference genes. A geometric mean was applied in that formula to use two reference genes instead of one (Vandesompele et al., 2002Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A and Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:research0034.; Hellemans et al., 2007Hellemans J, Mortier G, De Paepe A, Speleman F and Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:R19.) (^{Figure S1} Figure S1 - Mathematical model used for calculation of normalized expression quantities (NRQ) with two reference genes in qRT-PCR. ). The arithmetic means of Cq values obtained from second instar samples (larval stage analysis) and newly emerged workers (adult stages analysis) were used as calibrator.

Statistical analyses

Variation in expression levels of mfe and jhe among the distinct periods of late larval development (five biological replicates in each category; n = 30) was tested by the statistical model of Analysis of Variance (ANOVA). Moreover, a Linear Regression Analysis was made for both genes to test if the relation between expression levels and larval stages could fit into a line or curve. Polynomial terms were included when patterns were curvilinear. Larval periods were considered an ordinal variable in these analyses. The relation between mfe or jhe expression and age-related division of labor among workers (nine biological replicates in each category; n = 27) was tested using ANOVA, followed by Tukey HSD test to identify differences between each group pair. Relative expression ratios were transformed into logarithmic values prior to statistical analysis in order to increase residual homogeneity of variance and normality. The significance of statistical tests was considered at the 0.05 level. All statistical analyses were made with the software R version 3.6.3 (R Core Team, 2020R Core Team (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, https://www.R-project.org
https://www.R-project.org... ).

Results

First, we confirmed the identity of mfe and jhe transcripts amplified in M. interrupta. The mfe sequence shared a high identity level with the orthologous genes in other bees, especially in the closely related species M. quadrifasciata (97%) and M. scutellaris (97%), but also in the bumble bee Bombus terrestris (86%), and in A. mellifera (81%). The jhe sequence was almost identical to its ortholog in M. quadrifasciata (99%), and also shared a high percentage of identity with the ones in M. scutellaris (98%), in the stingless bee Scaptotrigona depilis (97%), and in A. mellifera (86%).

Then, we asked if the expression of two genes related to JH metabolism varies in relation to five physiological and behavioral subdivisions of late larval development in M. interrupta (Table 1): the penultimate instar (“L2”); the progressive feeding periods of the last larval instar (“L3.1”; “L3.2”; “L3.3”); and the two periods within cocoon-spinning stage (larvae pre-defecating “LPD”; defecating “LD”). Melipona bees, unlike honey bees, are not raised in brood cells of different sizes (Hartfelder et al., 2006Hartfelder K, Makert GR, Judice CC, Pereira GAG, Santana WC, Dallacqua R and Bitondi MMG (2006) Physiological and genetic mechanisms underlying caste development, reproduction and division of labor in stingless bees. Apidologie 37:144-163.), thus it was not possible to discriminate the sex and caste of individuals at the larval stages.

We found that expression levels of both mfe and jhe varied significantly among the larval stages (ANOVA for mfe: F = 3.35, p = 0.02; ANOVA for jhe: F = 8.67, p = 8.3x10^-5). Variation in mfe levels through late larval development could be best defined by a cubic polynomial regression (Cubic Regression Model: r² = 0.26, p = 0.01). The mfe expression increased at the transition from the penultimate to the last instar (L2 and L3.1), decreased while larvae were progressively feeding (L3.2 and L3.3), and started to increase again at the defecating period of the cocoon spinning phase (LD) (Figure 1A). Variation in jhe expression could be best defined by a parabola (Quadratic Regression Model: r² = 0.25, p = 0.007). In contrast to mfe, the jhe expression increased during the progressive feeding periods (L3.2 and L3.3), and gradually decreased during the cocoon-spinning phase (LD, especially) (Figure 1B).

Figure 1
‒ Relative expression levels of mfe (A) and jhe (B) through late larval development in Melipona interrupta. Each point represents an individual (n = 30). The sex and caste of the individuals could not be identified at that stage. The x-axis shows the larval stages (see Table 1 for legends). The y-axis shows the relative expression ratios (i.e., fold changes) of target genes in experimental samples relative to calibrator samples. The mean of Cq (quantification cycle) values obtained from the second instar samples was used as calibrator. (A) Variation in mfe expression can be described by a cubic polynomial regression (Cubic Regression Model: r² = 0.26, p = 0.01). (B) Variation in jhe expression can be described by a parabolic curve (Quadratic Regression Model: r² = 0.25, p = 0.007).

Next, we investigated if mfe and jhe expression is related to age-related division of labor among workers. We analyzed the expression in three groups: individuals within the first hours after emergence (newly emerged); workers that were carrying out activities within the nest (nurses); and workers that were performing tasks outside the nest (foragers).

We found that expression levels of mfe were altered when workers started to perform tasks (ANOVA: F = 31.81, p = 1.8x10^-7). mfe expression was significantly upregulated in nurses (Tukey test: diff = 1.74, p = 3x10^-5) and foragers (diff = 2.45, p = 2x10^-7) in comparison to newly emerged workers (Figure 2A). However, there was no significant change in mfe expression at the transition from nursing to foraging activities (diff = 0.71, p = 0.08). Expression levels of jhe, in turn, showed major differences at the nurse stage (ANOVA: F = 23.3, p = 2.39x10^-6). jhe expression was significantly upregulated from newly emerged to nurse stage (Tukey test: diff = 1.95, p = 3x10^-6), and downregulated from nurse to forager stage (diff = 1.58, p = 7x10^-5). No significant difference in jhe expression was found between newly-emerged and forager workers (diff = 0.37, p = 0.44) (Figure 2B).

Figure 2
‒ Relative expression levels of mfe (A) and jhe (B) in relation to age-related division of labor among workers of Melipona interrupta. Each point represents an individual (n = 27). The x-axis shows the groups of workers performing distinct activities. The y-axis shows the relative expression ratios (i.e., fold changes) of target genes in experimental samples relative to calibrator samples. The mean of Cq (quantification cycle) values obtained from the newly emerged workers was used as calibrator. Mean values of relative expression ratios are shown in gray. Expression of both mfe and jhe varied significantly among worker groups (ANOVA for mfe: F = 31.81, p = 1.8x10^-7; ANOVA for jhe: F = 23.3, p = 2.39x10^-6). (A) mfe expression increased significantly in nurses (Tukey test: diff = 1.74, p = 3x10^-5) and foragers (diff = 2.45, p = 2x10^-7) compared to newly emerged workers. However, there was no significant difference in mfe expression between nurse and forager stages (diff = 0.71, p = 0.08). (B) jhe expression increased significantly from newly emerged to nurse stage (Tukey test: diff = 1.95, p = 3x10^-6) and decreased significantly from nurse to forager stage (diff = 1.58, p = 7x10^-5). There was no significant difference in jhe expression between newly emerged and forager stage (diff = 0.37, p = 0.44).

Discussion

Expression of mfe and jhe suggests a role in modulation of JH levels during larval development of M. interrupta

Here we observed that the patterns of variation in gene expression throughout late larval instars were distinct between mfe and jhe genes in M. interrupta. The differences were more pronounced in the progressive feeding periods (L3.2 and L3.3) and pre-defecating larvae (LPD), when the relative expression of mfe was low and the relative expression of jhe was high.

Recent data report fluctuations in JH titers obtained for larvae of M. scutellaris in the same stages listed here (L2 to LD) (Cardoso-Júnior et al., 2017Cardoso-Júnior ACM, Silva RP, Borges NA, Carvalho WJ, Walter SL, Simões ZLP, Bitondi MMG, Vieira CU, Bonetti AM and Hartfelder K (2017) Methyl farnesoate epoxidase (mfe) gene expression and juvenile hormone titers in the life cycle of a highly eusocial stingless bee, Melipona scutellaris. J Insect Physiol 101:185-194.). It was observed that JH titers decrease at the transition to the last instar (L2 to L3.1), remain low during the subsequent stages (L3.2, L3.3 and LPD), and increase at the defecating period of the cocoon spinning phase (LD). The increase of mfe expression in LD observed in our study corresponds positively to the JH levels described for M. scutellaris. Also, the high levels of jhe expression observed in progressive feeding stages, as well as its decrease in LD stage, are inversely related to JH levels in M. scutellaris. Thus, the variation observed in the expression of both genes, especially of jhe at the transition to the defecating period of the cocoon-spinning phase, is compatible with the changes in JH titers observed in M. scutellaris.

Changes in JH (and also in ecdysone levels) at the transition to the last larval instar and the feeding period of holometabolous insects are critical for the determination of adult body size (Truman and Riddiford, 2002Truman JW and Riddiford LM (2002) Endocrine insights into the evolution of metamorphosis in insects. Annu Rev Entomol 47:467-500.; Emlen and Allen, 2003Emlen DJ and Allen CE (2003) Genotype to phenotype: Physiological control of trait size and scaling in insects. Integr Comp Biol 43:617-634.). Also, endocrine changes at the cocoon-spinning (defecating period) and prepupal stage (not analyzed here) are crucial for determining the final size of adult organs and body parts. These are the periods where the structures that will form adult body parts, which are called imaginal discs, grow by cell proliferation (Emlen and Allen, 2003Emlen DJ and Allen CE (2003) Genotype to phenotype: Physiological control of trait size and scaling in insects. Integr Comp Biol 43:617-634.). Experiments with JH measurements in the honey bee Apis mellifera and in the stingless bee Scaptotrigona postica depilis have demonstrated that these two periods are critical for the differentiation between queens and workers, in which JH titers are several folds higher in queen destined larvae than in workers (Rembold, 1987Rembold H (1987) Caste specific modulation of juvenile hormone titers in Apis mellifera. Insect Biochem 17:1003-1006.; Rachinsky et al., 1990Rachinsky A, Strambi C, Strambi A and Hartfelder K (1990) Caste and metamorphosis: Hemolymph titers of juvenile hormone and ecdysteroids in last instar honeybee larvae. Gen Comp Endocrinol 79:31-38.; Hartfelder and Rembold, 1991Hartfelder K and Rembold H (1991) Caste-specific modulation of juvenile hormone III content and ecdysteroid titer in postembryonic development of the stingless bee, Scaptotrigona postica depilis. J Comp Physiol B 160:617-620.).

In honey bees, it has been established that the critical window for caste-specific JH action is the transition from fourth to fifth instar, which is the last and longest instar in these bees (Dietz et al., 1979Dietz A, Hermann HR and Blum MS (1979) The role of exogenous JH I, JH III and Anti-JH (precocene II) on queen induction of 4.5-day-old worker honey bee larvae. J Insect Physiol 25:503-512.). Changes in JH titers at this period overlap with the nutritional switch from royal jelly to worker jelly in worker larvae. Consequently, queen larvae grow much larger in overall body size than workers during the feeding period (Hartfelder and Emlen, 2012Hartfelder K and Emlen DJ (2012) Endocrine control of insect polyphenism. In: Gilbert LI (ed) Insect Endocrinology. Academic Press, London, pp 464-522.). In stingless bees (non-Melipona species), queen larvae receive two or three times more food than workers, which makes them have an extended feeding period and higher JH levels. As expected, these queens also have a larger body size than workers (Hartfelder et al., 2006Hartfelder K, Makert GR, Judice CC, Pereira GAG, Santana WC, Dallacqua R and Bitondi MMG (2006) Physiological and genetic mechanisms underlying caste development, reproduction and division of labor in stingless bees. Apidologie 37:144-163.).

In Melipona bees, however, nutritional signals are not determinant for caste divergence. Actually, queens have a smaller body size before mating (Kerr, 1950Kerr WE (1950) Genetic determination of castes in the genus Melipona. Genetics 35:143-152.; Hartfelder et al., 2006Hartfelder K, Makert GR, Judice CC, Pereira GAG, Santana WC, Dallacqua R and Bitondi MMG (2006) Physiological and genetic mechanisms underlying caste development, reproduction and division of labor in stingless bees. Apidologie 37:144-163.). The critical period for caste determination in Melipona is the cocoon-spinning period (Hartfelder and Emlen, 2012Hartfelder K and Emlen DJ (2012) Endocrine control of insect polyphenism. In: Gilbert LI (ed) Insect Endocrinology. Academic Press, London, pp 464-522.). It has been demonstrated that JH application at the beginning of the cocoon-spinning period is sufficient to induce the development of Melipona queens with the same morphological (head/thorax size, eye width and hind leg structure) and reproduction-related traits (ovaries morphology, spermatheca volume and tergal glands patterns) as the natural ones (Campos et al., 1975Campos LAO, Velthuis-Kluppell FM and Velthuis HHW (1975) Juvenile hormone and caste determination in a stingless bee. Naturwissenschaften 62:98-99.; Campos, 1978Campos LAO (1978) Sex determination in bees. VI. Effect of a juvenile hormone analogue in males and females of Melipona quadrifasciata (Apidae). J Kansas Entomol Soc 51:228-234.; Bonetti, 1984Bonetti AM (1984) Efeitos do hormônio juvenil no desenvolvimento ovariano de Melipona quadrifasciata. Rev Bras Biol 44:509-516.; Bonetti et al., 1994Bonetti AM, Cruz-Landim C and Kerr WE (1994) Sex determination in bees. XXX. Effects of juvenile hormone on the development of tergal glands in Melipona. J Apic Res 33:11-14.). The variation patterns observed for JH titers in M. scutellaris (Cardoso-Júnior et al., 2017Cardoso-Júnior ACM, Silva RP, Borges NA, Carvalho WJ, Walter SL, Simões ZLP, Bitondi MMG, Vieira CU, Bonetti AM and Hartfelder K (2017) Methyl farnesoate epoxidase (mfe) gene expression and juvenile hormone titers in the life cycle of a highly eusocial stingless bee, Melipona scutellaris. J Insect Physiol 101:185-194.) and mfe and jhe genes in M. interrupta here are in accordance with these data from earlier studies. Taken together, those findings support the hypothesis that mfe and jhe expression plays a role in JH titers modulation during a critical moment for caste determination in M. interrupta.

Expression of mfe and jhe in M. interrupta adult workers indicates a complex role of JH in the social behavior of stingless bees

Here we found evidence that both mfe and jhe are upregulated in nurses, but only jhe is downregulated at the transition from within-hive to foraging tasks in workers of M. interrupta. This might be an evidence that JH titers are higher in foragers than in nurses in M. interrupta, which would be in accordance with what is known for honey bee workers.

In honey bees, it has been demonstrated that JH acts together with the major egg yolk protein, Vitellogenin (Vg), to coordinate the transition from nest bees to foragers (Amdam and Omholt, 2002Amdam GV and Omholt SW (2002) The regulatory anatomy of honeybee lifespan. J Theor Biol 216:209-228.). Hemolymph levels of Vg are inversely correlated to the JH titers: Vg is lower in foragers than in nurses (Rutz et al., 1976Rutz W, Wille LGH and Scher ML (1976) The function of juvenile hormone in adult worker honeybees, Apis mellifera. J Insect Physiol 22:1485-1491.; Fluri et al., 1982Fluri P, Lüscher M, Wille H and Gerig L (1982) Changes in weight of the pharyngeal gland and haemolymph titres of juvenile hormone, protein and vitellogenin in worker honey bees. J Insect Physiol 28:61-68.). In honey bee workers, Vg is not synthesized to be used for egg production, since they rarely lay eggs in the presence of a queen (Robinson and Vargo, 1997Robinson GE and Vargo EL (1997) Juvenile hormone in adult eusocial Hymenoptera: Gonadotropin and behavioral pacemaker. Arch Insect Biochem Physiol 35:559-583.). Rather, it acts as an endocrine factor (Nelson et al., 2007Nelson CM, Ihle KE, Fondrk MK, Page RE and Amdam GV (2007) The gene vitellogenin has multiple coordinating effects on social organization. PLoS Biol 5:e62.). This regulatory role of Vg and its relation with JH in honey bee workers are opposite to the general norm in insects, which states that JH plays a key role in the reproductive physiology of females (i.e., gonadotropic hormone) by stimulating Vg synthesis (Hartfelder, 2000Hartfelder K (2000) Insect juvenile hormone: From “status quo” to high society. Braz J Med Biol Res 33:157-177.). Thus, it has been proposed that the reproductive regulatory network was co-opted for the control of age polyethism during the evolution of honey bees (Page et al., 2012Page RE, Rueppell O and Amdam GV. (2012) Genetics of reproduction and regulation of honeybee (Apis mellifera L.) social behavior. Annu Rev Genet 46:97-119.).

In the stingless bees M. scutellaris, hemolymph levels of both JH and Vg are significantly lower in foragers than in nurse bees (Dallacqua et al., 2007Dallacqua RP, Simões ZLP and Bitondi MMG (2007) Vitellogenin gene expression in stingless bee workers differing in egg-laying behavior. Insectes Soc 54:70-76.; Cardoso-Júnior et al., 2017Cardoso-Júnior ACM, Silva RP, Borges NA, Carvalho WJ, Walter SL, Simões ZLP, Bitondi MMG, Vieira CU, Bonetti AM and Hartfelder K (2017) Methyl farnesoate epoxidase (mfe) gene expression and juvenile hormone titers in the life cycle of a highly eusocial stingless bee, Melipona scutellaris. J Insect Physiol 101:185-194.). Interestingly, egg production by nurse workers is common in many stingless bees. Workers can lay two types of eggs: trophic eggs, which are eaten by the queen; and reproductive eggs, which generate haploid males (Bourke, 1988Bourke AFG (1988) Worker reproduction in the higher eusocial Hymenoptera. Rev Lit Arts Am 63:291-311.). Thus, Cardoso-Júnior et al. (2017Cardoso-Júnior ACM, Silva RP, Borges NA, Carvalho WJ, Walter SL, Simões ZLP, Bitondi MMG, Vieira CU, Bonetti AM and Hartfelder K (2017) Methyl farnesoate epoxidase (mfe) gene expression and juvenile hormone titers in the life cycle of a highly eusocial stingless bee, Melipona scutellaris. J Insect Physiol 101:185-194.) hypothesized that JH does not play a role in age polyethism of stingless bees, but rather that it kept its ancestral gonadotropic function. These authors also pointed out that JH regulates worker reproduction in bumble bees (Bloch et al., 2000Bloch G, Borst DW, Huang Z-Y, Robinson GE, Cnaani J and Hefetz A (2000) Juvenile hormone titers, juvenile hormone biosynthesis, ovarian development and social environment in Bombus terrestris. J Insect Physiol 46:47-57.), which is the sister group of stingless bees in the phylogeny of corbiculate bees (Kapheim et al., 2015Kapheim KM, Pan H, Li C, Salzberg SL, Puiu D, Magoc T, Robertson HM, Hudson ME, Venkat A et al. (2015) Genomic signatures of evolutionary transitions from solitary to group living. Science 348:1139-1144.), and suggested that its gonadotropic function was retained in the Bombini/Meliponini clade.

Nevertheless, there are still many open issues regarding possible JH roles in worker behavior of stingless bees. First, stingless bees do not present significant differences from honey bees in their age-related division of labor (Kerr and Santos-Neto, 1956Kerr WE and Santos-Neto GR (1956) Contribuição para o conhecimento da bionomia dos Meliponini 5. Divisão de trabalho entre as operárias de Melipona quadrifasciata Lep. Insectes Soc 3:423-430.). Second, egg-laying by workers is a highly variable feature among species of stingless bee. There are workers that do not produce eggs in the presence of the queen; workers are capable of laying trophic, but not reproductive, and vice-versa; workers that lay reproductive eggs only occasionally; and workers that contribute to most of the male production (Tóth et al., 2004Tóth E, Queller DC, Dollin A and Strassmann JE (2004) Conflict over male parentage in stingless bees. Insectes Soc 51:1-11.; Hartfelder et al., 2006Hartfelder K, Makert GR, Judice CC, Pereira GAG, Santana WC, Dallacqua R and Bitondi MMG (2006) Physiological and genetic mechanisms underlying caste development, reproduction and division of labor in stingless bees. Apidologie 37:144-163.). The physiological regulation of that process must be flexible also. Furthermore, JH titers were measured in only one species of stingless bee. As pointed out by Cardoso-Júnior et al. (2017Cardoso-Júnior ACM, Silva RP, Borges NA, Carvalho WJ, Walter SL, Simões ZLP, Bitondi MMG, Vieira CU, Bonetti AM and Hartfelder K (2017) Methyl farnesoate epoxidase (mfe) gene expression and juvenile hormone titers in the life cycle of a highly eusocial stingless bee, Melipona scutellaris. J Insect Physiol 101:185-194.), JH measurement is a time and labor consuming method that is carried out only in a few laboratories, which has been a caveat to expanding this research to other stingless bees.

Taken together, our results raise the possibility that JH might play a role in the age-related division of labor in workers of M. interrupta and that JH function could be variable among stingless bees’ species, or even that JH levels might have more complex patterns and play different functions in age-defined workers within the same species. Our data still need support from JH measurements though, since other regulatory checkpoints after gene transcription could alter JH levels. Still, giving the diversity of behaviors displayed by workers of stingless bees, JH role in adult workers of eusocial bees must be a complex issue and further studies are required.

Expression patterns of mfe and jhe are variable among highly eusocial bees

Furthermore, our results demonstrated that gene expression of both mfe and jhe is significantly variable among the stages of the last larval instar, especially at the cocoon-spinning phase, which is critical for caste determination in M. interrupta. In A. mellifera, it has been demonstrated that expression of jhe, but not of mfe, plays an important role in the control of JH titers at larval stages. Mackert et al. (2008Mackert A, Nascimento AM, Bitondi MMG, Hartfelder K and Simões ZLP (2008) Identification of a juvenile hormone esterase-like gene in the honey bee, Apis mellifera L. - Expression analysis and functional assays. Comp Biochem Physiol Part B Biochem Mol Biol 150:33-44.) found that the variation in jhe expression is in accordance with the fluctuations in JH titers during the last larval instars of A. mellifera. Later on, Bomtorin et al. (2014Bomtorin AD, Mackert A, Rosa GCC, Moda LM, Martins JR, Bitondi MMG, Hartfelder K and Simões ZLP (2014) Juvenile hormone biosynthesis gene expression in the corpora allata of honey bee (Apis mellifera L.) female castes. PLoS One 9:e86923. ) observed that jhe expression and JHE activity are significantly higher in workers than in queens at the transition from fourth to fifth instar, when caste-specific differences in JH titers are at a maximum in A. mellifera. These same authors noticed that neither mfe expression nor that of other JH biosynthesis genes is significantly different between workers and queens at that period.

We also found that gene expression of mfe and jhe is variable among distinct behavioral stages of adult workers in M. interrupta. However, only jhe is regulated at the transition from within-hive to foraging tasks in workers. In M. scutellaris, Cardoso-Júnior et al. (2017Cardoso-Júnior ACM, Silva RP, Borges NA, Carvalho WJ, Walter SL, Simões ZLP, Bitondi MMG, Vieira CU, Bonetti AM and Hartfelder K (2017) Methyl farnesoate epoxidase (mfe) gene expression and juvenile hormone titers in the life cycle of a highly eusocial stingless bee, Melipona scutellaris. J Insect Physiol 101:185-194.) observed that mfe expression did not vary between nurses and foragers, which is in accordance with our results. In A. mellifera, however, the regulation patterns were opposite to our results. Bomtorin et al. (2014Bomtorin AD, Mackert A, Rosa GCC, Moda LM, Martins JR, Bitondi MMG, Hartfelder K and Simões ZLP (2014) Juvenile hormone biosynthesis gene expression in the corpora allata of honey bee (Apis mellifera L.) female castes. PLoS One 9:e86923. ) found that the expression of mfe and five other JH pathway genes are upregulated in foragers, while jhe expression was not significantly different between nurses and foragers. Thus, transcriptional regulation of jhe and mfe in larval and adult stages is variable between honey bees and Melipona bees, which suggests that mechanisms for regulation JH titers within the context of social regulation might not be conserved among bees.

In summary, our study provides support for mfe and jhe expression as important regulation points of JH levels at moments of larval development that are critical for caste determination in M. interrupta. The caveat in the identification of workers and queens in larval stages needs to be overcome for a better understanding of these issues. Interestingly, we found that regulation of jhe, but not mfe, is related to the nurse-forager transition in workers of M. interrupta. Moreover, our findings also suggest that highly eusocial bees have different mechanisms for regulating JH levels. Further investigations are required to provide deeper insights into the regulatory gene network that has evolved in the stingless bees group.

Acknowledgements

This research was finantially supported by the Governo do Estado do Amazonas, Secretaria de Estado de Desenvolvimento Econômico, Ciência, Tecnologia e Inovação (SEDECTI) and Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) - Editais POSGRAD (scholarship and financial grant), Universal Amazonas 030/2013 (financial grant) and PAPAC 005/2019 (publication fee support); and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Finance code 001 and Edital PVE 099/2012 (financial grant). DVB also received a scholarship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) as a PhD student of the Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva (PPG-GCBEv) of the Instituto Nacional de Pesquisas da Amazônia (INPA). Special thanks to Dr. Martin Hasselmann for his insightful comments and suggestions for the manuscript writing. Thanks also to the Centro de Apoio Multidisciplinar (CAM) of the Universidade Federal do Amazonas (UFAM) for the logistical support.

References

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Supplementary material

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Table S1 - Primers used in PCR (gene identification) and qRT-PCR (gene expression) for the analysis of mfe and jhe in Melipona interrupta.

Figure S1 - Mathematical model used for calculation of normalized expression quantities (NRQ) with two reference genes in qRT-PCR.

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