Dissertationen zum Thema „Cuticular Hydrocarbon“

Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Cuticular hydrocarbon (CHC) profiles of insects play roles in behavioural interactions within and between species, encompassing species-, colony- and mate-recognition. CHCs are largely genetically determined and are thus unique to each species, making them useful in chemotaxonomy. However, species exhibit intra-species variation in their CHC profile which can be the result of both intra-species genetic variation as well as environmental influences such as habitat effects, colony effects, diet, host switching, as well as adsorption of CHCs from other insects. Studies have found that the CHC profiles of a specific insect species will often exhibit variations between regions as well as the species of host the insect is associated with. Therefore, an ideal system to investigate the effects of genetic population structure and environment on the CHC profiles of insects is within the fig – fig wasp mutualism. Fig species occur in a wide variety of habitats and host a diverse complement of fig wasp species. We were therefore offered the opportunity to investigate a wide range of potential influences on fig wasp CHC profiles ranging from environmental to genetic effects. Firstly, through GC-MS we found that the CHC profiles of the fig wasps investigated are both species-specific and species-group-specific, with the species Elisabethiella glumosae, Elisabethiella stuckenbergi and Ceratosolen capensis, and two Otitesella species-groups (the Uluzi and Sesqui species-groups) separating out significantly. Consensus phylogenies (based on COI, Cytb and EF-1α) showed that within the galling fig wasp genus Otitesella there were multiple genetic lineages within a species-group which corresponds to species-level genetic variation, and that each genetic lineage was confined to a single host fig species. The CHC profiles reflected the genetic relationships between the two species-groups, and the CHC profiles within a species group could be differentiated by genetic lineage/host species. This indicated that although genetic lineage was mostly responsible for the observed variation in CHC profiles, factors associated with different host species also had an effect. Strong regional variation overriding both the influence of genetic lineage and factors associated with host species were observed in the CHC profiles of the fig wasps within a species-group. This regional variation in CHC profiles was also observed within two pollinating fig wasp species, Elisabethiella stuckenbergi and Ceratosolen capensis, which was not supported by population genetic data (COI and Cytb). In fact, very little genetic population structure was found within the pollinating species, even though the pollinators were collected across South Africa. The lack of genetic structure in pollinating fig wasps can be the result of high gene flow caused by the large dispersal capability of pollinating fig wasps. Our results indicated that fig wasp CHC profiles have the potential to be used in chemotaxonomy and are possibly used as species and mate-recognition cues by the fig wasps. Furthermore, we found both a regional and associated host species effect on the CHC profile. We suggest that the observed regional effect in this study could be attributed to habitat differences and differences in fig wasp community between regions. Moreover, the effect host species had on the CHC profiles may be as a result of dietary differences between galls in different host species. A possible consequence of the observed regional/host speciesassociated effect on fig wasp CHC profiles is that it could lead to pre-mating isolation within fig wasp species, which could ultimately result in speciation. In addition, our results indicated that the interpretation of the variation in the fig wasp CHC profile was dependent on the scale of the analysis: on a broad, inter-species-level scale, fig wasp CHC profiles were species-specific; on a finer intra-species scale, variation in CHC profiles occurred between fig wasps collected from different regions; and on a within-region scale, variation in CHC profiles within species-groups occurred between genetic lineages/host species. Future studies should look at the application of CHCs in chemotaxonomic studies on the fig wasp phylogeny, as well as the effect of fig wasp community composition on fig wasp CHCs.
AFRIKAANSE OPSOMMING: Kutikulêre koolwaterstof (KK) profiele van insekte speel rolle in die gedragsinteraksies binne sowel as tussen spesies, en behels die herkenning van spesieof kolonielidmaatskap asook potensiële maats. Kutikulêre koolwaterstowwe word meestal deur gene bepaal en is dus uniek vir elke spesie, wat dit handig maak vir chemotaksonomie. Spesies vertoon egter soms intraspesie variasie in hul KK profiele wat die gevolg kan wees van beide intraspesie genetiese variasie sowel as omgewingsinvloede soos habitat effekte, kolonie effekte, dieet, tussen-gasheer skuiwings, asook die adsorpsie van ander insekte se kutikulêre koolwaterstowwe. Studies het gevind dat die kutikulêre koolwaterstof profiele van ʼn spesifieke insek spesie op ʼn gereelde basis verskille vertoon tussen streke asook tussen die verskillende gasheer spesies waarmee die insek geassosieer is. Om hierdie redes is die vy – vy-wesp mutualisme ʼn ideale sisteem om die uitwerking van genetiese populasie struktuur en omgewing op die KK profiele van insekte te ondersoek. Vy spesies kom in ʼn wye verskeidenheid van habitatte voor en ondersteun ʼn diverse groep vy-wesp spesies. Dit het ons die geleentheid gebied om ʼn wye reeks moontlike invloede van vy-wesp KK profiele te ondersoek, van omgewings- tot genetiese invloede. Eerstens, deur die gebruik van GC-MS het ons gevind dat die KK profiele van die vy-wespe wat ondersoek was beide spesie-spesifiek en spesie-groep-spesifiek is, met die spesies Elisabethiella glumosae, Elisabethiella stuckenbergi en Ceratosolen capensis, asook twee Otitesella spesie-groepe (die Uluzi en Sesqui spesie-groepe) wat betekenisvol onderskei kon word. Konsensus filogenieë (gegrond op COI, Cytb en EF1-1α) het getoon dat daar in die gal-induserende vy-wesp genus Otitesella veelvuldige genetiese lyne binne die spesie-groepe voorgekom het ooreenstemmend met tussen-spesie genetiese variasie, en dat elke genetiese lyn beperk was tot ʼn enkele gasheer vy spesie. Die KK profiele het die genetiese verhoudings tussen die twee spesie-groepe weerspieël, en die KK profiele binne ʼn spesie-groep kon onderskei word op grond van hul genetiese lyn/gasheer spesie. Hierdie het getoon dat, alhoewel genetiese lyn meestal verantwoordelik was vir die waargeneemde variasie in KK profiele, faktore wat met verskille in gasheer spesies gepaard gaan ook ʼn effek gehad het. Sterk streeks-verbonde variasie wat beide die invloed van genetiese lyn, én faktore wat met verskille in gasheer spesie gepaard gaan, oortref het, was waargeneem in die KK profiele van die vy-wespe binne ʼn spesie-groep. Hierdie streeks-verbonde variasie in KK profiele was ook waargeneem in twee bestuiwende vy-wespe, Elisabethiella stuckenbergi en Ceratosolen capensis, ʼn resultaat wat nie ondersteun was deur die genetiese bevolkingsdata nie (COI en Cytb). In werklikheid was baie min genetiese bevolkings-struktuur opgespoor binne die bestuiwer spesies, selfs as was die bestuiwer spesies regoor Suid-Afrika ingesamel. Die tekort aan genetiese struktuur in die vy-wesp bestuiwers kan die gevolg wees van hoë geenvloei wat veroorsaak word deur die hoë verspreidingskapasiteit van bestuiwende vy-wespe. Die resultate toon aan dat vy-wesp KK profiele die potensiaal besit om in chemotaksonomie gebruik te word, en word moontlik deur vy-wespe gebruik as kenmerke vir die herkenning van spesie en potensiële maats. Verder was daar gevind dat daar beide ʼn streekseffek en ʼn effek geassosieer met gasheer spesie op KK profiele was. Ons stel voor dat die waargeneemde streekseffek in hierdie studie toegeskryf kan word aan verskille tussen habitatte asook streeksverbonde verskille tussen vy-wesp gemeenskappe. Boonop kan die effek wat gasheer spesie op die KK profiele gehad het ʼn gevolg wees van dieetverskille tussen die galle in verskillende gasheer spesies. ʼn Moontlike gevolg van die waargeneemde streeks/gasheer-spesie-geassosieerde effek op vy-wesp KK profiele is dat dit moontlik kon lei tot voor-paring-isolasie binne vy-wesp spesies, wat uiteindelik spesiasie kon veroorsaak het. Daarbenewens wys ons resultate dat die interpretasie van die variasie in die vy-wesp KK profiel was afhanklik van die skaal van die analise: op ʼn breë interspesie vlak was die vy-wesp KK profiele spesiespesifiek; op ʼn fyner intra-spesie vlak het variasie in KK profiele voorgekom tussen vy-wespe wat in verskillende streke ingesamel was; en op streeksvlak het variasie in die KK profiele binne spesie-groepe voorgekom tussen genetiese lyne/gasheer spesies. Toekomstige studies behoort te kyk na die toepassing van kutikulêre koolwaterstowwe in chemotaksonomiese studies van die vy-wesp filogenie, asook die effek wat vy-wesp gemeenskap samestelling het op vy-wesp kutikulêre koolwaterstowwe.

Diferenças no timing da melanização e esclerotização do exoesqueleto são evidentes quando se compara a morfologia externa de abelhas de hábitos sociais e as solitárias. A esta diferença convencionamos chamar de heterocronia da maturação cuticular, o termo heterocronia significando variações no tempo relativo, ou ritmo, de um evento ontogenético em relação ao ancestral ou entre taxons. Propusemos que as abelhas sociais, que após a ecdise permanecem na colônia por vários dias, alcançariam a maturidade de alguns sistemas orgânicos, entre eles o tegumento, muito mais tarde que as espécies de abelhas solitárias que ao emergir partem imediatamente para atividades extra-nidais. Neste contexto, o objetivo deste trabalho consistiu em testar esta hipótese utilizando o tegumento em maturação das espécies de abelhas sociais, Apis mellifera e Frieseomelitta varia, e da espécie solitária Centris analis, em estudos comparativos de expressão gênica, ultraestrutura e quantificação de hidrocarbonetos cuticulares (CHCs). Para isto utilizamos sequenciamento de mRNA (RNA-seq), microscopia eletrônica de transmissão (MET) e cromatografia de gás e espectrometria de massas (CG/MS). Os perfis de expressão de genes da via de melanização/esclerotização cuticular (ebony e tan) diferenciaram as espécies sociais da solitária, assim como a expressão de genes com função na via de metabolismo de quitina (Cda5, Idgf4 e chitooligosacchariodolytic-domain-like) e de genes codificadores de proteínas estruturais da cutícula (CPR14, CPR17, CPR18, CPR25, CPR23, CPR26, Apd-3 e Apd-like). Genes com função na regulação da maturação cuticular (FTZ-F1, E74, Hr46 e Hr4) se mostraram co-expressos nas espécies sociais e os perfis de expressão destes genes, exceto Hr46, e de outros reguladores (Ethr, Hr38, Rickets e Ptx-1) também diferenciaram as espécies sociais da solitária. Ressaltamos em nossas análises os genes do ciclo circadiano, cuja expressão tem relação com a deposição de quitina cuticular, além de genes de vias de pigmentação não melanínicas. As análises de MET, abrangendo outras três espécies de abelhas (Bombus brasilienses: primitivamente eussocial; Euglossa cordata: facultativamente social; Tetrapedia diversipes: solitária), mostraram diferenças consistentes entre a ultraestrutura e espessura das cutículas das espécies sociais e solitárias, o que reforçou nossos resultados de RNA-seq. A quantificação absoluta dos CHCs diferenciou as abelhas sociais da solitária, consistente com a hipótese de heterocronia da maturação cuticular e com os perfis de expressão de genes envolvidos na biossíntese de CHCs. Assim, além de desvendar transcriptomas de tegumento de três espécies de abelhas, a comparação da expressão gênica aliada à análise de ultraestrutura da cutícula e quantificação de CHCs levaram à caracterização de diferenças no processo de maturação cuticular entre as espécies sociais e solitárias
Differences in the timing of exoskeleton melanization and sclerotization processes are evident when comparing the external morphology of social and solitary bee species. Such differences may constitute a relevant example of cuticular maturation heterochrony, this term referring to a genetic change in timing of an ontogenetic process relative to an ancestor or between taxons. We proposed that social bees, which remain protected inside the colony for many days before initiating outside nest activities, would reach the maturity of some organic systems, such as the integument (epidermis and cuticle), later than solitary bees, which start such activities immediately after ecdysis. We tested this hypothesis in a comparative study of the developing integument of eusocial bees, Apis mellifera and Frieseomelitta varia, and the solitary bee Centris analis. Using RNA-seq, we verified that the expression profiles of genes involved in cuticular melanization and sclerotization (ebony and tan), chitin deposition and organization (Cda5, Idgf4, chitooligosacchariodolytic-domain-like), and cuticle formation (CPR14, CPR17, CPR18, CPR25, CPR23, CPE26, Apd-3, Apd-like) were positively, correlated between the two eusocial species, but not between the eusocial and the solitary species. Some of the genes with roles in regulating exoskeleton maturation (FTZ-F1, E74, Hr46, Hr4) were co-expressed only in the eusocial species. The expression profiles of these genes (except Hr46) and other regulatory genes (Ethr, Hr38, Rickets, Ptx-1) were also positively correlated exclusively in the eusocial bees. We also highlighted the expression of genes involved in non-melanin pigment production and the expression of circadian rhythm genes that could be related to chitin layers deposition. Transmission electron microscopy analysis of the integument of the two eusocial and the solitary bee species, in addition to other three bee species (the primitively eusocial Bombus brasilienses; the facultatively social Euglossa cordata; the solitary bee Tetrapedia diversipes), showed differences in cuticle ultrastructure and thickness, thus supporting the RNA-seq data. In agreement with our hypothesis, CHC quantifications were consistent with the expression levels of genes involved in CHC biosynthesis, thus differentiating the superficial cuticle layer of the eusocial and solitary species. Together, the integument transcriptomes, ultrastructure, and CHC quantification allowed us to characterize differences in the timing of cuticle maturation in social and solitary bees

Section One: Total synthesis of a cuticular hydrocarbon from the cane beetle Antitrogus parvulus utilizing bismuth mediated reactionsThis section describes investigations of 1,5-stereocontrol bismuth(III) mediated coupling reactions between allylbromide and aldehydes and successful application of this coupling chemistry in the total synthesis of a cuticular hydrocarbon. The bismuth(III) mediated coupling reaction was investigated using chiral bromide [(E,2R)-5-bromo-2,4-dimethyl-pent-3-enoxy]methylbenzene 1.79 with benzaldehyde and butyraldehyde; 12:1 diastereoselectivity in favour of forming the 1,5-anti product was observed in both model studies. This bismuth(III) coupling chemistry has been successfully applied in the construction of (4S,6R,8R,10S,16S)-4,6,8,10,16-pentamethyldocosane 1.21 and (4S,6R,8R,10S,16R)-4,6,8,10,16-pentamethyldocosane 1.22. 13C NMR spectroscopic comparison of the synthetic diastereomers with the natural product 1.17 revealed that (4S,6R,8R,10S,16S)-4,6,8,10,16-pentamethyldocosane 1.21 was consistent with the natural product. Therefore, it was concluded that the natural product 1.17 possesses the same relative stereoconfiguration as (4S,6R,8R,10S,16S)-4,6,8,10,16-pentamethyldocosane 1.21. Section Two: Towards the total synthesis of vioprolide DThis section describes attempts towards the total synthesis of vioprolide D. Initial disconnection revealed three tripeptide fragments. Fragment one benzyl (2R)-2-[[(2S)-2-[[(2S)-3-[tert-butyl(dimethyl)silyl]oxy-2-triisopropylsilyloxy-propanoyl]amino]propanoyl]amino]-4-methyl-pentanoate 2.26 was synthesized from commercial amino acid building blocks. Attempts to synthesize fragment two (S)-(9H-fluoren-9-yl)methyl 2-(((2S,3R)-1-(((S)-1-(allyloxy)-3-methyl-1-oxobutan-2-yl)(methyl)amino)-3-hydroxy-1-oxobutan-2-yl)carbamoyl)pyrrolidine-1-carboxylate 2.40 were unsuccessful because of a intramolecular cyclization reaction which led to the formation of piperazinedione 2.50, and this process stopped peptide elongation. Literature search showed that this side reaction could be prevented by changing the allyl ester group in dipeptide (S)-allyl 2-((2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tert-butyldimethylsilyl)oxy)-N-methylbutanamido)-3-methylbutanoate 2.42 to a tert-butyl ester group. Synthesis of thiazoline containing fragment three (E)-allyl 2-((R)-2-((S)-1-((2-nitrophenyl)sulfonyl)pyrrolidin-2-yl)-4,5-dihydrothiazole-4-carboxamido)but-2-enoate 2.57 was initially attempted via a cysteine cyclization reaction with an adjacent amide bond, this approach was unsuccessful due to extensive epimerization observed. In the modified synthetic strategy, thiazoline containing (E)-allyl 2-((R)-2-((S)-1-((R)-2-((tert-butoxycarbonyl)amino)-4-methylpentanoyl)pyrrolidin-2-yl)-4,5-dihydrothiazole-4-carboxamido)but-2-enoate 2.73 was successfully constructed as a single diastereomer via dehydration reaction of thioamide with adjacent serine residue using diethylaminosulfur trifluoride. The successful synthesis of tetrapeptide 2.73 would not only lead to the total synthesis of vioprolide D, but also potential access to other vioprolide analogues.

The evolution of isolating mechanisms within a species population impedes gene flow. This allows isolated populations to diverge along different trajectories, which may ultimately lead to the formation of new species. Our attempts to understand the evolution of isolating barriers have benefited enormously from studies of divergent populations that are still recognized as members of the same species. The co-occurrence of five acoustically distinct populations of the bushcricket Mecopoda elongata in south India provided us with the opportunity to study one such divergence of sympatric populations of a single species. In sympatric populations that share identical ecology, sexual selection has the potential to play a prominent role in the maintenance of reproductive isolation. Based on a previous traditional morphometric study, Mecopoda elongata in India were thought to be a morphologically indistinguishable cryptic species complex. The lack of morphological divergence suggests a less significant role of ecology in the divergence of the group. One possibility is that songtypes may be maintained by the preference of Mecopoda elongate females for mating with a specific songtype. In this thesis I show that female phonotaxis to their ‘own’ call has the potential to contribute to behavioural isolation among the songtypes and in particular between two songtypes with overlapping temporal call parameters. This finding is supported by an independent no-choice mating experiment utilizing the same two songtypes. To investigate the cues other than song that Mecopoda elongata females’ may use to exercise preference for their own type, I examined the composition of cuticular lipids in the cuticle and the detailed structure of secondary sexual characters. I was able to differentiate all Mecopoda elongata songtypes with high probability based on CHC profiles and geometric morphometrics of the sub genital plate and cerci. My study reveals that divergence in sexual traits other than acoustic signals, although dramatically less obvious in nature, is present among Mecopoda elongata populations. This provides potential mechanisms for premating isolation among Mecopoda elongata songtypes in the wild suggesting that reproductive isolation is maintained by female preferences for male sexual signals. Additionally, I discovered a parasitoid Tachinid fly responsible for infecting three different songtypes of Mecopoda elongata, namely Double Chirper, Two Part and Helicopter. This Tachinid fly appears to have specialized hearing organ to track down calling Mecopoda elongata males throwing light on potential selection pressure and possible mechanism for Mecopoda elongata song divergence.

Orientadores: Woodruff Whitman Benson, Jose Roberto Trigo
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Este trabalho discute alguns aspectos da ecologia da formiga lava-pés Solenopsis invicta e seus parasitóides, as moscas do gênero Pseudacteon (familia Phoridae), e da taxonomia de S. invicta e S. saevissima, ambas nativas da América do Sul. A alometria de colônias de S. invicta no sudeste do Brasil foi estudada através de medições das dimensões do ninho acima do solo, seguidas por escavação e medida da profundidade, biomassa e número de operárias da colônia, assim como da largura média da cabeça das operárias. Análise de regressão dos dados para 28 colônias forneceu novas relações alométricas que podem ser usadas para estimar o volume do ninho (acima e abaixo da superfície do solo), a biomassa de formigas, e o número de operárias a partir do volume do ninho acima do solo. A biomassa de formigas por unidade de volume do ninho, o número de operarias por unidade de volume do ninho, o peso médio por operária, e a largura média da cabeça das operárias foram essencialmente independentes do tamanho da colônia. Em contraste, estas quatro quantidades aumentam com o tamanho da colônia entre colônias de S. invicta na América do Norte. O ciclo sazonal e abundância de moscas parasitóides do gênero Pseudacteon na vizinhança de colônias de S. invicta no sudeste do Brasil foi observado ao longo de um período de 3 anos em li áreas de gramíneas nos arredores de Campinas. O número médio N de forídeos atraídos para um ninho de formigas lava-pés ao longo de um período padrão de observação (15 min) foi usado como uma medida da abundância de forídeos. As dimensões do ninho e algumas variáveis meteorológicas foram também registradas em cada ninho de formigas lava-pés. Pseudacteon tricuspis foi a espécie mais comum (70.4%), seguido por P. curvatus (23.8%), P. obtusus (1.9%) e P.litoralis (3.9%). As características mais notáveis das flutuações sazonais da abundância de Pseudacteon são picos abruptos com duração aproximada de um mês (valores máximos de N entre 4 e 10 forídeos/ninho), que ocorreram em abril e junho de alguns anos; para comparação, valores típicos de N estavam entre 0.2 e 2 forídeos/ninho. As populações de forídeos parecem ser destruídas por incêndios e inundações, mas recuperam-se em uma escala de tempo da ordem de meses. Nenhuma correlação significativa foi encontrada entre a abundância de Pseudacteon e variáveis meteoro lógicas, densidade, ou volume de ninhos de S. invicta, sugerindo que a abundância de Pseudacteon não está diretamente ligada a abundância de S. invicta. Flutuações quase idênticas em abundância de Pseudacteon foram observadas ao longo de um período de 6 meses em duas áreas de estudo separadas por cerca de 3 km, o que pode ser causado por dispersão de forídeos e conseqüente uniformização de densidades sobre áreas de vários quilômetros quadrados. Os números de forídeos atraídos pelos vários ninhos de S. invicta incluídos neste estudo seguiram uma distribuição de Poisson em baixas abundâncias de Pseudacteon (N < 0,5 forídeos/ninho); a distribuição tornava-se mais e mais contagiosa com o aumento da abundância de Pseudacteon (até o máximo observado, de N = 10 forídeoslninho). Esta observação sugere que a atratividade de um ninho de formigas lava-pés aos parasitóides aumenta com o número de forídeos que foram previamente atraídos para o ninho. Critérios quantitativos eficientes foram desenvolvidos para distinguir entre S. invicta e S. saevissima, que são morfológicamente muito similares e cujas distribuições geográficas na América do Sul se sobrepõe significativamente. Estes critérios resultaram da análise de 68 amostras de formigas lava-pés de 9 localidades espalhadas pelo estado de São Paulo (sudeste do Brasil) através de cromatografia gasosa e espectrometria de massa. 22 hidrocarbonetos cuticulares e 18 alcalóides piperidínicos foram identificados nas amostras estudadas, e suas abundâncias relativas foram analisadas utilizando análises de componentes principais e de funções discriminantes. Estas análises mostraram uma separacão nítida entre as duas espécies em um plano com certas coordenadas (P,Q), que são funções das abundâncias relativas dos hidrocarbonetos cuticulares. As amostras provenientes de uma das localidades (São José do Rio Preto) têm uma composição de hidrocarbonetos que é claramente diferente de todas as outras amostras consideradas neste estudo, mas também apresentam algumas caracteristicas em comum com cada uma das duas espécies. Uma possibilidade é que esta população constitua um hibrido de S. invicta e S. saevissima. As duas espécies também exibem uma separação razoavelmente nítida em um plano com coordenadas (R$), que são funções das abundâncias relativas de alcalóides piperidínicos. Os resultados das identificações de espécies baseados nestes critérios de separação quimica concordam com os resultados baseados em um critério morfológico, a presença ou ausência do dente clipeal mediano. A população "anômala" de São José do Rio Preto é morfologicamente classificada como S. invicta
Abstract: This work discusses some aspects of the ecology of the fire ant species Solenopsis invicta and its phorid parasitoid flies ofthe genus Pseudacteon, and ofthe taxonomy of S. invicta and S. saevissima, both of which are native to South America. The allometry of S. invicta colonies in southeast Brazil was studied by measurement of mound (aboveground) dimensions, followed by excavation and measurement of the colony depth, biomass, number of workers, and mean worker head width. Regression anaIysis of data for 28 colonies yielded new allometric relationships which can be used to estimate nest volume (above and below ground), ant biomass, and number ofworkers ITom mound (aboveground) volume. Ant biomass per unit nest volume, number of workers per unit nest volume, mean worker weight, and mean worker head width were nearly independent of colony size. In contrast, these four quantities increase with colony size among S. invicta colonies in North America. The seasonal cycle of abundance of Pseudacteon phorid flies in the vicinity of S. invicta colonies in southeast Brazil was observed over a 3-year period in 11 grass-covered areas in the outskirts of Campinas. The average number N of phorids attracted to a fire ant nest over a standard observation period (15 min) was used as a measure of phorid abundance. Mound dimensions and meteorological variables were also recorded at each fIre ant nest. Pseudacteon tricuspis was the most common species (84.2%), followed by P. curvatus (12.6%), P. obtusus (2.7%) e P. litoralis (0.5%). The most remarkabIe featores of the seasonal fluctuations of Pseudacteon abundance are sharp month-Iong peaks (peak N values between 4 and 10 phorids/nest) which occurred in Abril and June of some years; for comparison, at other times N was typically between 0.2 and 2 phorids/nest. Phorid populations seem to be destroyed by rifes and floods, but recover on a time scale of months. No significant correlation was found between Pseudacteon abundance and meteorological variables, density, or volume of S. invicta nests, suggesting that Pseudacteon abundance is not determined by, and does not determine, the abundance of S. invicta. Almost identical fluctuations in Pseudacteon abundance were observed over a 6-month period in two study areas separated by about 3 km, which may be due to phorid dispersaI and consequent density uniformization over areas of several square kilorneters. The nurnbers of phorids attracted by the various S. invicta nests inc1uded in this study followed a Poisson distribution at 10w Pseudacteon abundances (N < 0.5 phorids/nest), becoming more and more contagious at high Pseudacteon abundances (up to N= 10 phorids/nest). This observation suggests that the attractiveness oftire ant nests to phorid parasitoids increase with the nurnbeer ofphorids which have been previously attracted to the nest. Efficient quantitative criteria were developed for distinguishing between S. invicta and S. saevissima, which are morphologically very similar and whose geographical ranges in South Arnerica significantly overlap. These criteria resulted from the analysis of 68 tire ant samples from 9 localities across São Paulo state (southeast Brazil) via gas chromatography/mass spectrometry. 22 cuticular hydrocarbons and 18 piperidine alkaloids were identified in the studied samples, and their relative abundances were analyzed using principal component and discrirninant function analyses. These analyses showed a sharp separation between the two species in a plane with certain coordinates (P,Q), which are functions of the relative abundances of cuticular hydrocarbons. The fire ant samples from one of the localities (São José do Rio Preto) had a hydrocarbon composition which was markedly different from ali other samples considered in this study, but had some features in common with each ofthe two species. One possibility is that this population constitutes a hybrid of S. invicta and S. saevissima. The two species also show a reasonably sharp separation in a plane with coordinates (R,S), which are functions of the relative abundances of piperidine alkaloids. The species identification results based on these chernical separation criteria agree with those based on a morphological criterion, the presence or absence of the median clypeal tooth. The "anomalous" population from São José do Rio Preto is rnorphologically c1assified as S. invicta
Doutorado
Ecologia
Doutor em Ecologia

Os hidrocarbonetos cuticulares têm importante papel no processo de reconhecimento dos membros da colônia de insetos sociais. Muitos estudos têm mostrado variações qualitativas e quantitativas nestes compostos entre os insetos adultos. Contudo, abordagens referentes à modulação do perfil destes compostos durante a formação da cutícula são escassas, e se restringem aos estágios larval de holometábolos e de ninfas de hemimetábolos. O principal objetivo dessa pesquisa foi caracterizar o perfil de hidrocarbonetos cuticulares e a expressão de genes potencialmente relacionados à sua biossíntese durante o processo de formação e maturação da cutícula adulta. Os perfis de hidrocarbonetos foram caracterizados por meio de GC/MS e mostraram diferenças quantitativas marcantes que significativamente discriminaram as cutículas pupal, adulta-farata e adulta. Em paralelo, sequências de enzimas que catalisam a desaturação (desaturases) ou elongação (elongases) de lipídeos, disponíveis no banco de dados do NCBI, foram utilizadas para o desenho de primers e estudo da expressão gênica por meio de RT-qPCR. Cinco genes de desaturases, e oito genes de elongases mostraram variação de expressão estatisticamente significante no tegumento de abelhas adultas em comparação com pupas e adultas-faratas. Testes de correlação entre os perfis de expressão gênica e de hidrocarbonetos cuticulares evidenciaram os genes potencialmente envolvidos com a biossíntese destes compostos para a formação e maturação da cutícula. Estes resultados corroboram a hipótese de que nos insetos sociais, a cutícula só amadurece completamente por ocasião do início da atividade de forrageamento. Associando estes dados a análises de evolução molecular das desaturases e elongases, pudemos sugerir as etapas da via de síntese de hidrocarbonetos catalisadas por estas enzimas, e assim eleger genes candidatos a futuro silenciamento mediado por RNA de interferência para pesquisa de função.
Cuticular hydrocarbons are important for recognition of nestmates in social insect colonies. Many studies have shown qualitative and quantitative variations in the cuticular hydrocarbons between adult insects. However, approaches on developmental profiles of these compounds during cuticle formation and differentiation are scarce, and restricted to larval stages of holometabolous and nymphs of hemimetabolous. The main objective of this work was to characterize the cuticular hydrocarbons profiles and the expression of genes potentially involved in the biosynthesis of these compounds during the synthesis and differentiation of the adult cuticle in the honeybee. The hydrocarbons profiles were characterized using GC/MS and showed remarkable quantitative differences, thus discriminating the pupal, pharate-adult and adult cuticles from each other. In parallel, we used annotated sequences of enzymes catalyzing lipid desaturation (desaturases) or elongation (elongases), available in NCBI data bank, for primers design and gene expression analysis using RT-qPCR. Five desaturase genes and eight elongase genes showed statistically significant expression changes in the integument of adult bees in comparison to pupae and pharate-adults. Correlation tests supported roles of some of the desaturase and elongase genes in hydrocarbons biosynthesis for incorporation into adult cuticle. In addition, these results go along with the hypothesis that in social insects the cuticle is just completed when the insect starts forager activity. Taken together, these data and an analysis on the molecular evolution of desaturases and elongases allowed suggesting the steps in the pathway of cuticular hydrocarbons biosynthesis that are catalyzed by these enzymes, and also allowed to elect candidate genes for further functional studies using gene silencing mediated by RNAi.

Cuticular hydrocarbons (CHCs) are found on the outer cuticle of all terrestrial arthropods. Although their primary function is in desiccation prevention, these compounds have also been shown to play a variety of roles in insect chemical communication, from species and sex recognition to providing cues of dominance and attractiveness. However, despite growing evidence of their versatility as cues, our knowledge of how CHCs are used in mating interactions is limited to Drosophila and field crickets. In this thesis I investigate the roles CHCs play in interactions at each stage of the mating process in the broad-horned flour beetle Gnatocerus cornutus. I assess the relative importance of CHCs in influencing male reproductive success and examine the complex interplay between different episodes of selection and the mechanisms of sexual selection acting on males. I use a combination of behavioural assays, experimental manipulations and gas chromatography. First, I identify the role of CHCs as cues of sperm competition risk and intensity, demonstrating how the presence of male-derived CHCs on the cuticles of virgin females elicits males to adjust their pre- and post-copulatory investment (chapter 2), by providing information on the state of their competitive environment. I then go on to look at the stability of CHCs as cues of sperm competition over time, finding that they are highly sensitive to environmental degradation (chapter 3) and do not persist in the habitat substrate of this species. Next, I investigate how male CHCs determine fighting and mating success. By estimating and comparing the strength and form of sexual selection imposed by male-male competition and female mate choice, I show that male CHCs are subject to strong antagonistic sexual selection (chapter 4). By experimentally manipulating male CHC profile, I then attempt to verify the selection gradients estimated for female choice 3 (chapter 5). However, my experimental manipulation fails to verify the importance of male CHCs for female mate choice. Finally, I explore the role of same-sex sexual behaviour (SSB) in determining male reproductive success (chapter 6). I find evidence to suggest that SSB may in fact be a form of aggression in its own right, and demonstrate that SSB and fighting may provide equivalent means for males to overcome female choice and secure a mating advantage. My results indicate that CHCs play key roles as chemical cues throughout the mating process and significantly impact male reproductive success. My thesis reveals the intricate nature of the relationships between mechanisms of sexual selection, alongside highlighting the need to consider both the social and physical environment when investigating the importance of chemical cues. I discuss the implications of these results for the evolution of male CHCs and how my findings can be used to further our knowledge of this field.

A integridade das sociedades de insetos depende da capacidade dos indivíduos em discriminar aqueles que não pertencem às suas colônias; essas interações de reconhecimento são mediadas por hidrocarbonetos presentes na camada de cera da cutícula e atuam como mensageiros que codificam informação sobre o meio interno (colônia) e externo (ambiente). Os hidrocarbonetos são produzidos por células dérmicas e o perfil desses compostos recebe influência tanto de fontes endógenas quanto exógenas e isso o torna suscetível às mudanças temporais. A caracterização desses compostos em insetos-praga é importante, pois provêm subsídios tanto para pesquisa básica como para o manejo e controle de pragas. Nesse contexto, o presente estudo abordou as variações dos hidrocarbonetos cuticulares em relação às subcastas morfológicas das operárias (condição endógena) e utilização do substrato foliar (variável exógena) na formiga-cortadeira Atta sexdens, conhecida como uma das principais pragas herbívoras da região Neotropical. Os compostos foram extraídos em solvente apolar hexano e as amostras foram analisadas em sistema de cromatografia a gás acoplado à espectrometria de massas (CG-MS). Foram identificados 30 hidrocarbonetos com cadeia carbônica entre 19 e 40 átomos distribuídos em três classes de compostos, sendo a dos hidrocarbonetos ramificados a mais representativa, especificamente os trimetialcanos. Foram encontradas variações significativas para todas as variáveis analisadas, sugerindo que as subcastas possuem assinaturas químicas semelhantes quanto à diversidade de compostos, mas com grandes diferenças na proporção relativa dos mesmos. Em relação ao substrato foliar, colônias que tiveram acesso ao mesmo recurso apresentam uma composição química cuticular mais semelhante em relação àquelas que tiveram acesso a um recurso diferente. Complementarmente, os testes comportamentais mostraram que, na presença de uma coespecífica intrusa, as operárias residentes são muito mais agressivas em relação àquelas que utilizaram outro tipo de substrato foliar.
The integrity of insect societies relies on the ability of individuals to discriminate between nesmates from non-nestmates. These interactions are mediated by hydrocarbons spread all over the cuticle that act as messengers coding information about the colony and external environment. Cuticular hydrocarbons are produced by dermic cells and its composition is influenced by both endogenous and exogenous sources which makes them subject to temporal changes. Thus, this study approached the variations on this chemical profile in relation to the worker subcastes and type of foliar substrate used by leafcutter ant Atta sexdens, this species is known as one of the major neotropical herbivore pests and this kind of study is important because it can provide subsides in researches related to both basic biology as to pest control methods. The compounds were extracted using apolar solvent (hexane) and the samples were analyzed using gas chromatography - mass spectrometry technique (GC-MS). It was found 30 hydrocarbons which carbon chains varies between 19 and 40 atoms separated in three classes of compounds and the branched hydrocarbons as the most abundant one, especially the trimethyalcanes. The statistical analysis revealed significant differences on all the variables analyzed suggesting that subcastes possess similar chemical signatures as to the variety of compounds but with great differences in relative proportions thereof. Furthermore, the results show that foliar substrate has influence on the composition of cuticular hydrocarbons and concomitantly affect the interspecific recognition, thus colonies that had access to the same substrate has similar cuticular composition comparing to those who do not. Additionally, behavior tests showed that in the presence of a conspecific intruder the resident workers tend to be much more aggressive in relation to those who had fed on a different foliar substrate.

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A vespa social basal Mischocyttarus nomurae Richards tem ocorrência exclusiva no Brasil e foi registrada nos estados do Ceará, Bahia e Minas Gerais. Na Bahia, ocorre em três municípios da Chapada Diamantina: Lençóis, Mucugê e Rio de Contas. Nesta última, foram estudadas populações com relação à sua biologia básica, buscando responder perguntas relacionadas aos seguintes aspectos: morfologia externa dos imaturos, perfis comportamental, morfofisiológico e químico, substituição de rainhas, arquitetura de ninhos e hábitos de nidificação. Com relação à morfologia dos imaturos, a taxa média de crescimento da larva foi de 1,48. Com exceção da larva de 1o ínstar, que revelou-se menor do que o ovo, a espécie apresentou o padrão do grupo: presença de lobos, estes em número de dois e projetados para a frente, só totalmente desenvolvidos quando a larva atinge o 5o instar; as mandíbulas das larvas de 5o instar apresentam um único dente alongado e o primeiro espiráculo é mais do que duas (3,1) vezes maior em diâmetro que os demais. As cápsulas cefálicas da larva de 5º ínstar e da pré-pupa não diferiram significativamente. Após os estudos dos perfis morfofisiológico, químico e comportamental, foi verificado que as fêmeas de M. nomurae não apresentam distinção em sua morfologia externa, mas cinco tipos de desenvolvimento ovariano foram observados, três menos desenvolvidos (A1, A2 e B) e dois mais desenvolvidos (C e D). Foram detectados 34 compostos na sua cutícula, cujas cadeias variaram de 18 a 33 átomos de carbono. Os compostos consistiram em alcanos lineares (9%), alcanos ramificados (89%) e alcenos (2%). Foram identificados três grupos de fêmeas: Operária 1, Operária 2 e Rainha e a análise discriminante do perfil dos hidrocarbonetos cuticulares relacionada a esses grupos de fêmeas apresentou um p-valor significativo (Wilks' lambda= 0,135, F= 1.665, p = 0,0227). Após oito dias da remoção da fêmea dominante do ninho, em todas as colônias, exceto uma, a fêmea era anteriormente a fêmea β. Fêmeas com ovários não desenvolvidos podem realizar comportamentos de fêmea dominante, assim como uma fêmea com ovário desenvolvido comportouse como forrageadora típica. Houve diferença entre algumas atividades exercidas pelas fêmeas posicionadas em 2º lugar no ranking (fêmea β) e as demais fêmeas do ninho (3ª posição em diante), antes e após a remoção experimental da rainha. M. nomurae apresenta um sistema de gerontocracia flexível na sua substituição de rainhas e suas fêmeas sucessoras tornam-se mais agressivas que as rainhas originais, assumindo tarefas de dominantes, mesmo sem estarem inseminadas. Como algumas espécies do gênero, a maioria das colônias apresentou hábitos de nidificação relacionados a ambientes antrópicos. Destes, 61,2% das colônias utilizaram material de origem vegetal (madeira e palha). A altura média de construção do ninho com relação ao solo foi elevada, semelhante as das demais espécies do gênero (2,20 ± 0,83, 0,45 – 5,00m). A maioria dos ninhos foi construída com o favo voltado para baixo (70,15%) e em substrato horizontal (53,73%). O único favo do ninho pode ser arredondado ou elíptico, raramente retangular. O número de camadas de mecônio variou de zero a cinco e a espécie M. nomurae apresenta ninhos pequenos, com suas colônias apresentando um baixo número de adultos produzidos, embora haja uma alta taxa de reutilização de células.
The basal social wasp Mischocyttarus nomurae Richards occurs exclusively in Brazil, having been recorded there in the states of Ceará, Bahia, and Minas Gerais. It is known to occur in three municipalities in the Chapada Diamantina mountains of Bahia State: Lençóis, Mucugê, and Rio de Contas. Population studies were undertaken in the latter municipality to address questions related to the basic biology of M. nomurae: the external morphologies of the immatures; their behavior; their morpho-physiological and chemical profiles; queen substitutions; nest architecture, and nidification habits. In terms of the morphologies of the immatures, the mean larval growth rate was 1.48. With the exception of the 1st instar larvae, which are smaller than the eggs, the species demonstrated the standard developmental pattern for the group: the presence of lobes (two) projected forward, those only becoming fully developed in the 5th instar stage; mandibles in the 5th instar stage with a single elongated tooth, and the first spiracle with a diameter more than two (3.1) times greater than the others. The cephalic capsules of the 5th instar larvae and pre-pupae do not significantly differ. Studies of the morpho-physiological, chemical, and behavioral profiles of those wasps indicated that the females of M. nomurae do not have distinct external morphologies, although five different types of ovarian development were observed: three less-developed types (A1, A2 and B); and two more developed types (C and D). Thirty-four distinct chemical compounds were detected in the cuticle, with carbon chains varying from 18 to 33 atoms. Those carbon compounds consisted of linear alkanes (9%), branched alkanes (89%), and alkenes (2%). Three groups of females were identified: Workers 1, Workers 2, and Queens. Discriminant analyses of the cuticular hydrocarbons of those female groups demonstrated a significant p-value (Wilks' lambda= 0.135, F= 1.665, p = 0.0227). Eight days after the removal of the dominant female from the nest, female substitutions were observed all of the colonies (except one) assuming the condition of a new queen (from the ranks of the β females). Females with undeveloped ovaries do not perform behaviors associated with dominant females, nor do females with developed ovaries behave as typical foragers. There were differences between some of the activities performed by second-rank females (β females) and other females in the same nest (from the 3rd position down), both before and after experimentally removing the queen. M. nomurae demonstrates a flexible gerontocratic system in terms of queen substitutions, and the new queens become more aggressive than the original queens, assuming dominant roles even without being inseminated. As with many species of the same genus, most of the colonies nidify in anthropically modified environments. Of those colonies, 61.2% built nests using plant material (wood and leaves). The nests were constructed at reasonably high mean heights above ground level (2.20 ± 0.83 m; from 0.45 – 5.00 m), similar to other species of the genus. Most of the nests were affixed to horizontal substrates (53.73%) and constructed with the comb facing downward (70.15%). A nest comb can be rounded or elliptic, rarely rectangular. The numbers of meconium layers varied from zero to five. The nests of M. nomurae are generally small, with their colonies producing only small numbers of adults - although with high rates of comb cell reutilization.

The Common bed bug, Cimex lectularius L., suddenly reappeared in developed countries in the past 15 years. The factor contributing to the sudden resurgence of the bed bugs is insecticide resistance. In this study, we investigated the mechanisms of reduced cuticular penetration type insecticide resistance in bed bugs. First, we determined and compared the lethal dosage (LD50) of a pyrethroid insecticide using topical and injection application. The resistant strain not only had significantly greater resistance ratios, but also demonstrated significantly greater penetration resistance ratios. This provided the evidence of the reduced cuticular penetration in bed bugs. Second, we determined the levels of gene transcription (CPR-type cuticle protein genes) using real-time quantitative polymerase chain reaction (qRT-PCR). We identified 62 putative bed bug cuticle protein-encoding contigs based on the presence of the Chitin-binding 4 (CB4) domain. Based on the qRT-PCR analysis of the mRNAs, we found many of the genes were up-regulated in the resistant strain suggesting thickening of the cuticle or increasing the cuticular proteins might be involved in the reduced cuticular penetration. Third, we identified and described the cuticular proteins using the matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometry (MALDI-TOF/TOF). The total of 265 peptides were identified, among which 206 belonged to one of 50 confidently identified proteins. We identified the CPRL, CPF, CPFL, TWDL, and CPAP1 family proteins. The profile of the cuticular proteins between the resistant and the susceptible strains bed bugs were almost identical. Fourth, we determined and compared the cuticular thickness using Scanning Electron Microscopy (SEM). We found statistical differences of the cuticular thickness among different strains (populations), however, correlation between the levels of insecticide resistance and cuticular thickness were not found. Finally, we identified and described bed bug cuticular hydrocarbon profiles using Gas-Chromatography and Mass-Spectrometry (GC-MS). The total of 87 compounds in addition to n-alkanes were extracted and identified. There were no correlation found with the concentration and the levels of insecticide resistance. However, several additional compounds exhibited the correlation between the concentration of the compounds and the levels of insecticide resistance. Overall, we found three lines of evidence to support reduced cuticular penetration as a mechanism of insecticide resistance in some bed bug populations. This study provides additional evidence of the reduced cuticular penetration type resistance in bed bugs.
Ph. D.

La gestion de la résistance aux insecticides et plus particulièrement aux pyréthrinoïdes chez les vecteurs du paludisme est un enjeu majeur pour conserver les acquis de la lutte antivectorielle contre le paludisme. Parmi les mécanismes de résistance, de nombreux travaux ont porté sur les mutations de la cible insecticides (kdr) ou encore les enzymes qui métabolisent les molécules insecticides. La résistance cuticulaire chez Anopheles gambiae a jusqu'à ce jour au mieux été suspectée mais pas démontrée. Dans ce cadre, le but de ce travail était donc de déterminer i) s’il y a une implication de la cuticule dans le phénotype résistant d’Anopheles gambiae aux pyréthrinoïdes ; ii) de déterminer les principaux acteurs de cette résistance. Nous avons construit une souche d’Anopheles gambiae présentant un phénotype résistant (MRS) mais ne portant pas la mutation kdr. Notre approche a été d’étudier l’expression des gènes, mais aussi celle des protéines de la cuticule et celles intervenant dans la détoxication. L’ultrastructure de la cuticule, sa composition en chaines hydrocarbonées ainsi que sa perméabilité à la deltaméthrine ont aussi été testés. Nous avons montré que la cuticule est impliquée dans le phénotype résistant plus particulièrement en réduisant significativement la pénétration de la deltaméthrine dans le corps de l’insecte. La structure même de la cuticule est profondément modifiée chez MRS. Nos résultats indiquent l’implication des membres de la famille CPAP3 dans la résistance cuticulaire et probablement des chaines hydrocarbonées. Des enzymes métaboliques sont également impliqués. Au cours de ce travail, nous avons démontré l’existence de la résistance cuticulaire chez An.gambiae, ce mécanisme agit de concert avec les mécanismes de détoxication. La caractérisation des différents acteurs nous permettra surement de trouver de nouvelles cibles pour la lutte anti-vectorielle.Mots clés : Résistance, cuticule, An.gambiae, deltaméthrine, ultrastructure, perméabilité, protéines
The management of Anopheles insecticide resistance, especially to pyrethroids, is a key challenge to preserve success of vector control against malaria. Among resistance mechanisms target site mutation (kdr) and metabolic resistance are well known. Cuticular resistance in malaria vectors has been overlooked, just suggested but not established so far. The aim of this PhD work was to demonstrate i) the involvement of cuticle in pyrethroid resistance in An. gambiae; ii) to determine which components act on this mechanism. To achieve this, we built An.gambiae strain resistant to pyrethroids and free of kdr mutation (MRS). We studied both cuticular and metabolic genes and proteins expressions by transcriptomic and proteomic approaches. Cuticle ultrastructure and biochemical composition were also investigated. At least, cuticle permeability to deltamethrin was also assessed. Our results showed that cuticle is involved in resistant phenotype in An.gambiae. MRS cuticle reduces insecticide uptake in the mosquito’s body linked to an increasing cuticle thickness in MRS. Expression and chemical studies revealed that CPAP3 family and epicuticular hydrocarbons takes part in this process. This work also suggested that metabolic enzymes act together with cuticular mechanism to resistant phenotype. How the different components interacts could improve our knowledge of resistance and bring new target for vector control.Keywords: Resistance, cuticle, An.gambiae, deltamethrin, ultrastructure, permeability, proteins

Nutrição, regulação da expressão gênica e estresse oxidativo são características corresponsáveis pelo desenvolvimento e tempo de vida. Muitos animais apresentam uma relação clara entre o aumento da longevidade e a diminuição da reprodução quando submetidos a restrições alimentares. Contudo, a relação nutrição-longevidade não está completamente elucidada em organismos prioritariamente inférteis e cuja alimentação varia durante a vida adulta, tais como abelhas operárias da espécie A. mellifera. Para explorar tais questões, operárias recém-emergidas foram confinadas em gaiolas e alimentadas com uma dieta isenta de proteína (DNP), ou uma dieta rica em proteínas (DP) por sete dias. Investigamos a influência das dietas sobre: a morfologia das glândulas hipofaringeanas, o transcriptoma do corpo gorduroso, o acúmulo de dano oxidativo, a composição de hidrocarbonetos cuticulares (CHC) e a sobrevivência. Operárias do grupo DNP apresentaram menor sobrevivência e menor grau de ativação das glândulas hipofaringeanas do que operárias do grupo DP. A anotação funcional do transcriptoma de operárias do grupo DNP revelou ainda a ativação da resposta a estímulos, diferenciação, migração e desenvolvimento celular e de projeções neuronais. Todas essas são características compatíveis ao que se observa em operárias naturalmente mais velhas, como é o caso das forrageiras. A anotação funcional do transcriptoma de operárias do grupo DP revelou a ativação do metabolismo de proteínas, lipídios e carboidratos e regulação do sistema imunológico, características ligadas a operárias naturalmente jovens, como as nutridoras. Um total de 436 genes codificadores de proteínas foi apontado pelo sequenciamento em larga escala como dieta-responsivos (fold change > 2). O sequenciamento de RNAs curtos revelou três miRNAs dieta-responsivos (fold change > 2), miR-31a, miR-100 e miR-125, todos superexpressos no grupo DNP. Dentre esses 439 genes codificadores (mRNAs) e não codificadores de proteínas (miRNAs), dez foram experimentalmente validados em corpos gordurosos por RT-qPCR e quatro dos dez revelaram um perfil de expressão semelhante em cérebros frente às dietas. Juntos, nossos achados sustentam a existência de um circuito biológico integrado entre cabeça e corpo gorduroso capaz de regular os diferentes aspectos do controle da longevidade e do comportamento social. O ensaio de estresse oxidativo revelou não haver diferença entre o acúmulo de danos em cabeças nos dois grupos alimentares, talvez sinalizando a existência de uma resistência ou defesa antioxidante mais acentuada no sistema nervoso, protegendo-o. Por outro lado, observamos um nível maior de estresse oxidativo em corpos gordurosos de operárias do grupo DNP, sugerindo um déficit da resposta antioxidante, característica atrelada ao envelhecimento. Referente aos perfis de CHC, esses são similares entre operárias do grupo DP e operárias jovens (maior proporção de n-alcanos), bem como entre operárias do grupo DNP e operárias velhas (maior proporção de alcenos). Em conjunto, nossos resultados indicaram que uma dieta livre de proteínas antecipa o envelhecimento e sugerimos alguns novos marcadores do status nutricional e da progressão do desenvolvimento adulto em operárias: XP_624408.2, XP_393528.3 e XP_006561863.1, os órtólogos de CG9986, CG6058, CG2852, CG32031, CG5848 e CG9331, CG11138, CG10160, CG2674, CG5178, CG15884 e CG1322, além dos três microRNAs já citados. As alterações relacionadas ao status nutricional e envelhecimento, observadas nesse trabalho, suportam o uso de A. mellifera como um excelente organismo modelo no campo da nutrigenômica, bem como contribuem para o entendimento das modulações promovidas pela dieta no desenvolvimento adulto desse organismo.
Nutrition, oxidative stress and gene expression regulation are features responsible for development and lifespan. Many animals have a well-established relationship between an increase in longevity and a decrease in reproduction when subjected to dietary restrictions. However, the biological circuit nutrition-longevity is not fully elucidated in organisms primarily infertile and whose food consumption varies during adulthood, such as honey bee workers A. mellifera. To explore such questions, newly-emerged A. mellifera workers were confined in cages and fed a high protein diet (DP) or a protein free diet (DNP) for seven days. We analyzed the morphology of hypopharingeal glands, mRNAs and miRNAs global expression, oxidative damage, cuticular hydrocarbons profiles (CHC), and workers survival. Workers from DNP group had lower survival and lower activation of hypopharingeal glands than workers from DNP group. The functional annotation of DNP group transcriptome revealed activation of response to stimuli, cell differentiation, cell migration, cell growth and development of neuronal projections. All these biological processes are consistent with naturally older workers, such the foragers. The functional annotation of DP group transcriptome revealed activation of protein, lipid and carbohydrate metabolism and regulation of the immune system, features linked to younger workers, such nurses. Large-scale sequencing revealed that 436 protein-coding genes are diet-responsive (fold change > 2). Small RNAs sequencing revealed that three miRNAs are diet-responsive (fold change > 2), miR-31a, miR-100 and miR-125, all of them overexpressed in DNP group. Among these 439 coding (mRNA) and non-coding (miRNA) genes, 10 were validated in fatty bodies by RT-qPCR, and four of them showed a similar expression profile in brains in response to diets. Altogether, our results support the existence of an integrated biological circuit between head and fat body, which regulates different aspects of lifespan control and social behavior. Oxidative stress assay showed no difference between damage accumulation in honeybees heads from DP and DNP groups; maybe supporting the existence of a sharp resistance or antioxidant defense in the nervous system, protecting it. On the other hand, we observed a greater accumulation of oxidative stress markers in fat bodies of DNP, suggesting a deficit of antioxidant response, an aging feature. The CHC profiles are similar between DP group workers and young workers (high proportion of n-alkanes), and also similar between DNP group workers and old workers (high proportion of alkenes).Taken together, our results suggest that protein-free diet anticipates aging process and provides new markers of nutritional status and progression of workers adult development: XP_624408.2, XP_393528.3, XP_006561863.1, the orthologs of CG9986, CG6058, CG2852, CG32031, CG5848 e CG9331, CG11138, CG10160, CG2674, CG5178, CG15884 and CG1322, besides the three microRNAs already mentioned. The genetic changes related to nutritional status and aging observed in this work support the use of A. mellifera as an excellent model organism in the field of nutrigenomics and also contribute to the understanding of modulations promoted by diet on the adult development of this organism.

O gênero Polistes é considerado um grupo modelo para a compreensão da sociobiologia das interações de dominância/subordinação em animais invertebrados, devido à pequena diferenciação de castas que resulta em um alto potencial de conflitos reprodutivos entre os membros da colônia. A construção de ninhos satélites e os seus efeitos na estrutura colonial de algumas espécies de Polistes, constituem um campo de estudo de interesse para a área da evolução do comportamento social. Neste estudo foram quantificados os padrões sazonais do desenvolvimento e a composição colonial, bem como a relação desses padrões com a variação climática sazonal. Foram também comparadas as relações que determinam a hierarquia de dominância em colônias de P. satan em relação a um modelo teórico proposto para uma hierarquia linear de dominância. Além disso, mostramos os padrões de interações agonísticas entre indivíduos que compõem a hierarquia e suas inter-relações em colônias não manipuladas e manipuladas (remoção da dominante). Foram também analisados padrões de comunicação visual (marcas de dominância) e química (hidrocarbonetos cuticulares). Os resultados mostraram que fêmeas dominantes em colônias em pré-emergência apresentaram padrões de pigmentação cefálica e perfis de hidrocarbonetos cuticulares significativamente distintos das fêmeas subordinadas ou substitutas. Os aspectos relacionados à comunicação química e visual e suas implicações biológicas são discutidas.
The genus Polistes is an organism model to understand the sociobiology in invertebrates. This group presents low caste differentiation that result in potencial reproductive conflicts between nestmates. The construction os satellite nests and their effects on the colonial structure in some Polistes are an interesting topic to the evolution of socail behavior. In this study we quantify the seasonal patterns of nest development and thei composition. We analyzed the relationship between these patterns and climatic variation. The aspects related to the hierarchy of dominance in P. satan were compared to a theoretical model that predicts a dominance hierarchy im equilibrium. In addition, we presented the agressive interaction between individuals composing a hierarchy in experimental (dominant removal) and control colonies. Patterns of both visual (dominance labels) and chemical communication (cuticular hydrocarbons) were also verified in P. Satan. Dominant females from pre-emergence colonies present both distinct patterns of cephalic pigmentation and cuticular hydrocarbons than other females. These aspects involved with the sociobiology of Polistes and their biological implications are discussed in this study.

Directional selection is prevalent in nature yet phenotypes tend to remain relatively constant, suggesting a limit to trait evolution. The genetic basis of evolutionary limits in unmanipulated populations, however, is generally not known. Given widespread pleiotropy, opposing selection on a focal trait may arise from the effects of the underlying alleles on other fitness components, generating net stabilizing selection on trait genetic variance and thus limiting evolution. Here, I look for the signature of stabilizing selection for a suite of cuticular hydrocarbons (CHCs) in Drosophila serrata. Despite strong directional sexual selection on CHCs, genetic variance differed between high and low fitness individuals and was greater among the low fitness males for seven of eight CHCs. Univariate tests of a difference in genetic variance were non-significant but have low power. My results implicate stabilizing selection, arising through pleiotropy, in generating a genetic limit to the evolution of CHCs in this species.

Mourella caerulea, popularmente conhecida como mirim-de-chão ou bieira, e Plebeia nigriceps, comumente chamada mirim nigriceps, são meliponíneos ocorrentes no sul do Brasil, polinizadores de plantas nativas e cultivadas. M. caerulea está principalmente relacionada ao bioma Pampa e seu hábito de nidificação é subterrâneo. P. nigriceps nidifica em frestas de rochas e muros, sendo encontrada tanto no Pampa como em Mata Atlântica. O presente trabalho teve como objetivo avaliar a variabilidade populacional destas duas espécies, através de três metodologias: a morfometria geométrica de asa, perfil de hidrocarbonetos cuticulares e sequenciamento de fragmentos de genes mitocondriais. Foram coletadas operárias de 24 colônias de M. caerulea em cinco localidades de sua distribuição natural e 53 colônias de P. nigriceps em oito localidades no estado do Rio Grande do Sul. Para análise do padrão de venação da asa, foram marcados 13 marcos anatômicos na asa anterior direita de cinco a 20 operárias por colônia. A análise genética foi realizada avaliando um fragmento do gene Citocromo Oxidase I em M. caerulea, e Citocromo B em P. nigriceps. A análise morfométrica demonstrou estruturação dos grupos com separação estatisticamente significativa (<0,0001) entre as localidades de M. caerulea. As distâncias morfométricas estão correlacionadas com a distância geográfica, e coerentes com regiões fisiográficas do bioma Pampa. A análise dos perfis de hidrocarbonetos distinguiu colônias de diferentes localidades, mas suas distâncias não apresentaram correlação com as distâncias geográficas. Foram encontrados seis haplótipos, todos exclusivos, com diversidade nucleotídica de 0,01631e uma diversidade haplotípica (Hd) de 0,74. Para P. nigriceps, as análises morfométricas apresentaram diferenças significativas entre localidades e correlação com as distâncias geográficas e biomas. A análise dos perfis de hidrocarbonetos distinguiu colônias de diferentes localidades, mas suas distâncias não apresentaram correlação com as distâncias geográficas. Foram encontrados 17 haplótipos, todos exclusivos, com diversidade nucleotídica de 0,0147 e diversidade haplotípica (Hd) de 0,94. A presença de diversos haplótipos exclusivos, perfis morfométricos e de hidrocarbonetos cuticulares em populações pertencentes a diferentes biomas indicam a necessidade de uma atenção especial para estas populações no momento da definição de estratégias de conservação das espécies. Uma especial atenção às abelhas da espécie Mourella caerulea, que além de ser uma espécie representante única de um gênero monoespecífico, apresenta grandes distâncias populacionais entre os indivíduos de todas as localidades amostradas.
Mourella caerulea and Plebeia nigriceps are two stingless bees with occurrence in the South region of Brazil. The first is commonly known as mirim-de-chão or bieira and the second is known as mirim nigriceps. Both species are important pollinators of native flora and crops. M. caerulea is related to Pampa biome and place nests on the ground. P. nigriceps occurs in both Pampa biome and Atlantic Rain Forest. This thesis had the objective of to evaluate the population variability of these species through three techniques: geometric morphometrics of wing, cuticular hydrocarbons (CHC) profiles and sequencing of fragments from mitochondrial DNA genes. We collected workers from 24 colonies for M. caerulea from five localities, and 53 colonies of P. nigriceps from eight localities in Rio Grande do Sul State. For the geometric morphometrics analysis, we used 13 landmarks plotted in the right forewing of five to 20 workers per nest. The fragments of mtDNA genes used for the molecular approach were from Cytochrome Oxidase I for M. caerulea and Cytochrome B for P. nigriceps. The morphometric approach discriminated the populations of M. caerulea from different localities (<0,0001). The morphometric distances are correlated to geographic distances and go along with the physiographic regions of Pampa biome. CHC profiles differentiated the colonies of M. caerulea from different localities, but chemical distances are not in agreement with geographic distances. We found six haplotypes (all exclusives) with a nucleotide diversity of 0.01631 and a haplotype diversity (Hd) of 0.74. For P. nigriceps, morphometric analysis was significant separating localities and in accordance with the geographic distances and biomes. CHC distinguished the colonies, but there was no significant correlation between this result and the geographic distances or biomes. mtDNA revealed 17 haplotypes (all exclusives) with a nucleotide diversity of 0.0147 and a haplotype diversity (Hd) of 0.94. The discovery of different exclusives haplotypes, the morphometric and CHC profiles when comparing population belonging to different biomes indicate that we need to give a particular attention for these species at the moment of create conservation strategies for both biomes from Rio Grande do Sul. M. caerulea deserves a special concern once it is the only species of the monospecific genera, and its populations are distant between themselves.

Forensic entomology relies on accurate identification of forensically important blowflies to species level, preceded by ageing the Calliphorid specimens present on a cadaver to determine the post-mortem interval (PMI). The task of identifying blowflies based on morphologial criteria can be challenging due to complex keys, limited diagnostic features in the immature stages and poor preservation of entomological samples. Therefore, the larvae will mostly be reared to adult flies to confirm identification, which is a time consuming process. This thesis presents work examining the cuticular hydrocarbon (CHC) profiles of three forensically important blowfly species in the UK, Lucilia sericata, Calliphora vicina and Calliphora vomitoria with two main aims to this study. Firstly to establish if CHC analysis could be used to determine whether the three species yield characteristic profiles, allowing for identification to be achieved. The second aim was to examine the hydrocarbon profiles over time to determine if chemical changes occurred at certain points in time, giving an indication of age. The CHCs were extracted from all life stages (empty egg cases, larvae, pupae, empty puparial cases and adult flies) and analysed using gas chromatography – mass spectrometry (GC-MS) and Direct-analysis-in-Real-Time MS (DART-MS). Statistical interpretation was applied in the form of principal component analysis (PCA) and preliminary Artificial Neural Networks (ANN). Results showed species-specific characteristics within the chromatograms in all life stages, meaning distinctions can be made between the three species, even in 1st instar larvae. Significant chemical changes were observed within the hydrocarbon profiles over time, hence accurate ageing could be established for larvae, empty puparial cases and adult flies. Early results show great potential to utilise this technique and to develop it into a highly useful identification and ageing tool.

Chez les insectes sociaux, la capacité à reconnaître les compagnons du nid des autres a évolué du fait de l’intérêt à distinguer les individus apparentés. En effet, il est avantageux que les actes de coopération soient uniquement dirigés vers les individus apparentés. La faculté de discrimination repose sur la capacité à percevoir les signaux de reconnaissance (hydrocarbures cuticulaires) d'un individu rencontré et de le comparer avec une représentation de l'odeur de sa propre colonie. L'ontogénèse de la reconnaissance des compagnons du nid et les processus d'apprentissage qui pourraient les sous-tendre ont été peu explorés jusqu’à ce jour. Il a été suggéré que les insectes sociaux apprennent l'odeur de leur colonie pendant les premières étapes de la vie adulte. L'objectif de ce travail était de rechercher les principales caractéristiques de l'ontogénèse du processus de reconnaissance avec une approche comparative, en utilisant deux modèles : les fourmis et les guêpes sociales. Nous avons montré que le mécanisme général de reconnaissance proposé pour le genre Polistes ne s'appliquait pas à la guêpe P. dominula. Nous avons étudié les mécanismes alternatifs, suggérant l'apprentissage pré-imaginal et le mécanisme d'auto-référence. Nous avons mis en évidence pour la première fois que la fourmi Aphaenogaster senilis apprenait, durant la vie larvaire, des signaux l'importance possible de processus cognitifs précédemment négligés tels que chimiques importants pour la reconnaissance. Ainsi, l’existence et la fonctionnalité de la mémoire larvaire ont été révélées chez les fourmis, élargissant nos connaissances sur les mécanismes de reconnaissance des compagnons du nid, l'apprentissage et la mémoire
In social insects the ability to recognize nestmates from non‐nestmates evolved as a result of the advantages of distinguishing related individuals. Indeed, are gained only if cooperative acts are directed towards realtives. This discrimination ability relies on the capacity of perceiving recognition cues (cuticular hydrocarbons) of an encountered individual and of comparing them with a neural representation of the own colony odour. Relatively little is known about the ontogeny of nestmate recognition, and the learning processes that might be involved. It has been suggested that social insects learn their colony odour during the early stages of adult life. The overall aim of the present work was to investigate the main features of the ontogeny of nestmate recognition process with a comparative approach, using two different models: ants and social wasps. In particular, I investigated wheter general recognition mechanism proposed for the Polistes genus applies to Polistes dominula and I found that this is not the case. I studied alternative mechanisms and provided novel insights into the ontogeny of nestmate recognition in social wasps, suggesting the possible importance of previously neglected cognitive processes, such as pre-imaginal learning and self-reference phenotype matching. In ants, I showed for the first time that Aphaenogaster senilis learn chemical cues important for adult nestmate recognition during the larval life, thus larval memory is something present and functional in ants. This result gives significant insight for the study of nestmate recognition, the mechanisms at the basis, learning and memory in ants, opening new questions concerning interesting phenomena as pre-imaginal learning

Sexual conflict occurs when the fitness interests of males and females do not align with one another. The resolution of sexual conflict often depends on the level of control each sex has on the behavior in conflict. In Pogonomyrmex harvester ants with a genetically determined caste system, two separate lineages interbreed with one another during summer mating swarms. Diploid offspring sired by a single lineage develop into reproductive queens, while offspring sired by opposite-lineage parents develop into sterile workers. This results in sexual conflict, as males which mate with opposite lineage queens will produce only workers, resulting in no fitness benefit, while queens must mate with opposite-lineage males in order to obtain workers and survive. Despite these fitness differences, males do not discriminate between lineages prior to mating. One possible reason for the lack of male discrimination is that queens "mask" their identity cues, making discrimination difficult for males. In eusocial insects, identity cues are encoded by cuticular hydrocarbons (CHC’s) found on the exoskeleton of the insects. These cues contain information on the insect's reproductive status, sex, species, state, and nest membership. In addition to their communication functions, CHC’s also serve as desiccation-resistance molecules, preventing water from freely passing out of the cuticle. However, molecules that are best-suited for communication functions are poor desiccation resistance molecules, and molecules that are best-suited for waterproofing lack the diversity needed for communication; therefore, a tradeoff between these two functions is expected. In this dissertation, I explore sexual conflict in these ants and the chemical recognition cues that likely play a role in this conflict. To test for cryptic strategies in harvester ant mating swarms, I experimentally paired males and females from two interbreeding lineages of harvester ant with different fitness outcomes based on pairing, and measured the propensity to initiate copulation, pre-copulatory time, time in copula, and rate and amount of sperm transferred in each mating. Although females controlled copulation duration, males altered sperm transfer rates, resulting in no quantitative difference in total sperm transfer between lineages. To test for thermal constraints on the diversity and composition of cuticular hydrocarbon profiles, and changes in CHC profiles that occur in workers isolated from the queen, I surveyed the cuticular hydrocarbon profiles of a species complex of harvester ants. The CHC profiles of ants from more xeric environments showed evidence of constraints, while isolated workers differentiated from their queen-raised sisters, although not in queen-specific molecules. To test for queen identity masking and lack of discrimination ability in mating swarms, I tested for convergence in the CHC profiles of reproductives in two hybridizing lineages in response to the sexual conflict playing out in this species. Differences in CHC profiles were lost during the mating swarm, likely limiting male ability to discriminate between mates, limiting discrimination ability in mating swarms. To study the genetic regions that control CHC production, I created a physical linkage map of two of the interbreeding populations, and used that map to perform quantitative trait loci analysis on the cuticular hydrocarbon profile of recombinant males. One significant region associated with 13-methylnonacosane contained numerous odorant receptor genes, suggesting a link between that CHC production and the receptors that detect it, while a second region associated with n-pentacosane contained numerous genes that control expression levels. Overall, the genetic caste determination system in these ants leads to antagonistic coevolution between species. This coevolution is likely reinforced by the thermal constraints and exchange of recognition cues between species, lowering the ability of useful discrimination between lineages during mating swarms.

What processes contribute to the evolution of reproductive isolation and the coexistence of interfertile species in the same habitat? This thesis investigates the relative roles of species interactions and intraspecific processes in contributing to reproductive isolation. I combine behavioural and genomic approaches to test hypotheses about what mechanisms maintain the general species boundary between two closely related field cricket species: Teleogryllus oceanicus and T. commodus. These species are a classic study system for sexual communication and readily hybridize in the laboratory, however little is known about species interactions in sympatric populations. I examine patterns of geographic variation in two key sexual traits: calling song and cuticular hydrocarbons (CHCs), and the geographic distribution of genetic variation across a broad sample of allopatric and sympatric populations. I test whether X chromosomes play a pronounced role in population divergence and reproductive isolation. Using close range mating trials and hybridization experiments I identify numerous pre-mating and post-mating barriers between the species. The results indicate that the species are currently reproductively isolated and the pattern of population differentiation does not strongly support contemporary species interactions contributing to phenotypic diversity. Numerous barriers exist between the species, in particular hybrid females are sterile in both cross directions, while hybrid males are relatively fertile. This provides a rare exception to Haldane's rule which is central to many genetic theories of speciation. Established theory predicts that X chromosomes should play a pronounced role in the evolution of both pre- and postzygotic barriers. Contrary to this, I found no evidence that X chromosomes contribute to hybrid female sterility. Moreover, X-linked loci exhibited an unexpected pattern of reduced population differentiation within species, but increased species divergence compared to autosomal loci, which may indicate selective sweeps or sex-biased processes. Taken together, the results suggest that the causes and consequences of X chromosome evolution, in particular among XO taxa, may contradict some of the established theories.

One of the major research challenges is the ability to test selective forces in a wild population. A recent discovery of a new dipteran species, Protopiophila litigata, can enable researches to test selection in the wild. Most research has focused on mating behaviour, male mating success and senescence. In this study a small sample of wild mating and non-mating flies were collected, cuticular hydrocarbons were extracted and morphometric traits were obtained to assess the strength of sexual selection. There was significant linear sexual selection on cuticular hydrocarbons and, mid tibia length, hind tibia length and wing length. Overall, further establishes P. litigata as a model species for studying selection in the wild. Earlier studies have demonstrated strong sexual selection on male cuticular hydrocarbons in Drosophila serrata. Recently wing interference patterns have been documented to be under sexual selection in Drosophila melanogaster. A sample of cuticular hydrocarbons and wing interference pattern values were analyzed to understand the combined effects on male mating success. Cuticular hydrocarbons were under sexual selection, however wing interference patterns were not. Overall, this study confirms selection on cuticular hydrocarbons, but highlights the difficulty in accurately capturing and measuring wing interference patterns.

Over the last 100 years sexual selection has advanced into a vast field of theoretical and empirical research. While Darwin’s idea of female preference being an integral mechanism of sexual selection is no longer debated, our understanding of female preference is still very limited. For example, we know little about the genetic variation in female preference, and the costs of preference over and above the costs of mating with particular male phenotypes. Additionally, while costs of mate choice are well documented, the benefits of mate choice and their implications are still debated. For example, controversy exists over the inevitability of good gene benefits and their capability to promote adaptive sexual selection. Furthermore, the adaptiveness of sexual selection itself is debated. Our understanding of the traits involved in mate choice is also far from complete. Here I investigated aspects of sexual selection in Drosophila simulans, employing a range of behavioural approaches along with artificial selection and environmental manipulations. The findings presented here indicate that female preference can evolve when directly selected on, and that preference itself is not particularly costly. There was also no conclusive evidence for the good genes benefits of mate choice in D. simulans. These benefits are considered crucial in promoting the adaptiveness of sexual selection, and although we found sexual selection to be adaptive under some test conditions it was not adaptive in other conditions. Our investigations into traits involved in mate choice established sex-specific genetic variation in cuticular hydrocarbons and the genetic architecture of this trait was found to sex-specific evolution of cuticular hydrocarbons under natural and sexual selection. Additionally, we found that a secondary sexual character, the sex combs was positively allometric – just like most signalling and weapon traits, and there was no association between trait fluctuating asymmetry and trait size. These findings collectively indicate that sexual selection in D. simulans is consistent with classical models of this process.

La fourmi d'Argentine (Linepithema humile) est une des espèces invasives parmi les plus nuisibles au monde. Dans le cadre des invasions biologiques, les résistances biotiques sont souvent les dernières barrières des écosystèmes. Une hypothèse, jamais testé in natura, de résistance de la part d'une fourmi du genre Tapinoma face à L. humile a été émise. Pour le démontrer, nous avons choisi d'expérimenter en laboratoire et in natura. Nos terrains d'études privilégiés ont été, outres des sites du littoral méditerranéen français, 4 îles situées en face du littoral envahi par la fourmi d'Argentine. Parmi celles-ci, deux contiennent à la fois la fourmi d'Argentine et des espèces du genre Tapinoma. L'identification difficile des espèces du genre Tapinoma étant sujette à une controverse scientifique, nous avons dû différencier de façon certaine cinq d'entre elles, par leurs morphologies et leurs signatures chimiques. En Provence, trois espèces ont été observées : T. erraticum, T. madeirense et T. nigerrimum. Sur l'île de Ratonneau, un suivi sur 2 ans de la vitesse d'invasion de L. humile en milieu naturel, avec ou sans T. nigerrimum, met en évidence une vitesse d'invasion très faible, voire un recul dans la zone à T. nigerrimum. Les colonies de T. nigerrimum de l'île de Ratonneau semblent faire obstacle à la progression de l'invasion. Des études comportementales préliminaires, conduites en laboratoire, entre fragments de nids de T. nigerrimum et L. humile nous avaient permis de confirmer les capacités de T. nigerrimum à résister face à l'invasive. Ainsi, les ouvrières T. nigerrimum défendent mieux leurs territoires, allant jusqu'à accaparer celui des L. humile
The Argentine ant (Linepithema humile) is one of the world's worst invasive species. We used various analyses to determine its invasion pattern on the Provence coast, confirming the presence of a second supercolony. Chemical and behavioral analyses of worker interactions on the supercolony border identified a peaceful border zone. When biological invasion occurs, biotic resistance is often ecosystems' last defense. Could a dominant ant from Tapinoma genus resist L. humile invasion? To demonstrate this possibility, we conducted both laboratory and in natura experiements. Besides the invaded Mediterranean coast, we selected four nearby islands, two of which were found to contain both L. humile and Tapinoma spp. Identification of Mediterranean Tapinoma being controversial, we used both morphological and chemical analyses to ensure reliable discrimination. On the coast, we found T. erraticum, T. madeirense and T. nigerrimum. In natura, on Ratonneau Island, two-year field monitoring showed a slow rate of Argentine ant invasion and even retreat, faced with T. nigerrimum. T. nigerrimum colonies seem to block L. humile invasion. Laboratory behavioral studies between nests of T. nigerrimum and L. humile confirm T. nigerrimum's ability to stand up to Argentine ants: T. nigerrimum workers protect and defend their territories better, even taking over Argentine ant nests

Regulation of reproduction is one central feature of social life. In particular, only a few individuals are in charge of producing offspring in eusocial species. This division of the reproductive labour is mainly mediated by pheromones emitted by the queens in social insects. These queen pheromones may signal the presence of a fertile queen so that workers react accordingly by taking care of her and not reproducing.

Here I investigated two aspects of the reproduction of two polygynous ant species. The first one, Linepithema humile, is a unicolonial, highly polygynous and invasive species. It has been the focus of numerous studies about queen pheromones; in particular, it has been reported that queen cuticular hydrocarbons (CHC) profile is related to queen fertility. The other one, Tapinoma erraticum, is a multi-colonial, weakly polygynous and native species. Workers can lay haploid eggs in the absence of the queens which is impossible for Linepithema workers.

The major part of my thesis dealt with the queen fertility signalling issue. In the first two chapters I demonstrate the link between queen fertility and queen pheromone output. I first study a queen releaser pheromone, the queen retrieval behaviour. This behaviour is performed by the workers who lay a chemical trail toward a queen located outside the nest. I successfully show this behaviour to be related to queen fertility, and not mating status, in L. humile and T. erraticum since only fertile queens (mated or not) induce such recruitment. I then highlight the role of queen fertility in the prevention of worker reproduction in T. erraticum. Again, mated fertile queens and unmated fertile queens are both able to induce such primer effect. In a third chapter I report that CHC profiles may discriminate female castes (workers, queens, virgin queens, and virgin egg-laying queens) in T. erraticum. Finally, chapter 4 summarizes my attempts to prove that CHC may be involved in queen retrieval or queen attraction. None of the various bioassays tested allows me to demonstrate the putative role of CHC as queen pheromone.

In a second part, I was interested in the consequences of sex determination in T. erraticum. Chapter 5 presents the flow cytometry methodology (FC) which allowed me to score the number of sperm cells from spermathecae of several ant species, and to demonstrate that polygynous species (such as L. humile and T. erraticum) store less sperm than monogynous ones. FCM also allows determining the ploidy of sperm and adult somatic cells and chapter 6 which presents a large survey on the ploidy level within the species T. erraticum. This species displays diploid males that may produce diploid sperm which in turn can father a viable triploid female progeny. I report differences in the frequency of triploidy among female castes, the proportion of triploid workers being more important than triploid virgin queens whereas I never observed triploid mated fertile queens. Such results greatly suggest a putative regulatory mechanism involved in the rearing of triploid females. In the last chapter I investigated two populations that differ in the occurrence of triploid workers. I report these populations to vary in the number of queens and workers per nest.

/La régulation de la reproduction est un aspect essentiel de la vie sociale. En particulier, chez les espèces eusociales, seuls quelques individus sont impliqués dans la production de la descendance. Dans les sociétés d’insectes, une telle division du travail reproducteur est principalement assurée par l’émission de phéromones par les reines. Ces phéromones royales renseignent les membres de la colonie sur la présence d’une reine fertile, de telle sorte que les ouvrières réagissent en s’occupant d’elle et en s’abstenant de se reproduire.

Au cours de ce travail, je me suis intéressé à deux aspects de la reproduction au sein de deux espèces de fourmis polygynes. La première espèce, Linepithema humile, est invasive, unicoloniale et hautement polygyne dans les régions à climat méditerranéen. Elle a fait l’objet de nombreuses études portant notamment sur les phéromones royales. En particulier, il a été montré que le profil d’hydrocarbures cuticulaires (HCC) des reines est corrélé à leur fertilité. La seconde espèce, Tapinoma erraticum, est une espèce indigène, multi-coloniale et faiblement polygyne. Ses ouvrières sont capables de pondre des œufs mâles en absence de reines, ce dont sont incapables les ouvrières de Linepithema.

Je me suis principalement intéressé à la question de la signalisation de la fertilité des reines. Dans les deux premiers chapitres, je démontre le lien existant entre la fertilité des reines et la production de phéromone royale. J’ai d’abord étudié une phéromone incitatrice (releaser) qui provoque un recrutement royal. Ce comportement collectif très caractéristique correspond à la mise en place d’une piste chimique en direction d’une reine découverte par les ouvrières en dehors du nid. Je montre que ce comportement est lié à la fertilité de la reine chez les espèces L. humile et T. erraticum car seules les reines fertiles (fécondées ou non) sont capables de d’induire le recrutement royal. Je mets ensuite en évidence le rôle de la fertilité des reines dans la régulation de la reproduction des ouvrières de T. erraticum. A nouveau, les reines fécondées fertiles et les reines vierges fertiles sont toutes deux capables d’induire un même effet déclencheur (primer), en l’occurrence, l’inhibition de la reproduction des ouvrières. Dans un troisième chapitre, je montre que les profils d’hydrocarbures (HCC) permettent de distinguer sans ambiguïté les différentes castes femelles (ouvrières, reines fertiles, reines vierges et reines vierges pondeuses) chez T. erraticum. Enfin, le chapitre 4 résume mes tentatives pour démontrer le rôle des HCC dans les phénomènes de recrutement royal ou d’attractivité des reines. Aucun des bio-essais réalisés ne me permet de démontrer l’implication des HCC dans la phéromone royale.

Dans une seconde partie, je me suis intéressé aux conséquences du déterminisme du sexe chez T. erraticum. Le chapitre 5 présente cytométrie de flux (CF), une méthode qui me permet de compter les spermatozoïdes stockés dans les spermathèques de quelques espèces de fourmis et de montrer que les reines des espèces polygynes (telles que L. humile et T. erraticum) stockent moins de sperme que les espèces monogynes. La CF permet aussi de déterminer le niveau de ploïdie des cellules spermatiques ou somatiques chez l’adulte. Je me sers de cette application dans le chapitre 6 afin d’étudier le niveau de ploïdie au sein de l’espèce T. erraticum. Je montre que, dans les populations étudiées, il existe des mâles diploïdes et que ces mâles peuvent produire du sperme diploïde fertile, capable d’engendrer une descendance femelle triploïde. Je note des différences dans la fréquence des femelles triploïdes :la proportion d’ouvrières triploïdes est significativement plus importante que celle des reines vierges triploïdes. De plus, je n’ai jamais observé la présence de reines fécondées fertiles triploïdes. De tels résultats suggèrent fortement la présence d’un phénomène de régulation au cours de l’élevage du couvain triploïde. Dans le dernier chapitre, j’ai étudié deux populations de T. erraticum qui diffèrent au niveau de la proportion d’ouvrières triploïdes. Ces populations présentent des différences significatives dans le nombre de reines et d’ouvrières par nid.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Un extrait hexanique comportementalement actif est obtenu par rincage superficiel de la cuticule des femelles. La composition chimique de cette fraction est analysee par chromatographie et spectrometrie. Les composes a effet aphrodisiaque sont des hydrocarbures a longues chaines (37 carbones) satures et ramifies avec 2 radicaux methyl. Les constituants epicuticulaires des males ne depassent pas 31 atomes de c. Chez les larves des 2 sexes, les composes leges disparaissent en meme temps que se mettent en place les composes plus lourds. La pheromone sexuelle supposee chez les femelles de g. T. Est assez specifique. Cependant la specificite de reponse des males n'est pas etroite

Cuticular hydrocarbons (CHC) abound on the surface of arthropods. In spite of their simple structure (molecules of carbon and hydrogen atoms), they provide pivotal functions in insects: their hydrophobic properties confer the insects a means to regulate water balance and avoid desiccation, whereas their diversity has enhanced their use as signals and cues in a wide range of communication and recognition processes. Although the study of CHC in insects over the past two decades has provided great insight into the wide range of functions they play, there is still a gap in understanding how they diversify and evolve. In this thesis, I have used members of the family Chrysididae to explore patterns of diversification of CHC. Most of the species of cuckoo wasps in this study are specialized parasitoids or kleptoparasites of mainly solitary hymenopteran hosts. Other hosts of the family include butterflies or stick insects. Cuckoo wasps are a particular interesting model to study the evolution of cuticular hydrocarbons because of their chemical adaptations that allow them to remain unrecognized by their hosts. Chemical insignificance (the reduction of the total amount of CHC on the cuticle) and chemical mimicry (the de novo production of CHC profiles resembling those of their female host) have been described in some representatives of the family and unpublished evidence suggests chemical deception is widespread in Chrysididae (Chapter 2). Nonetheless, to trace the evolution of any trait of interest, a reliable phylogenetic reconstruction of the family is required. Therefore, the first study of this thesis constitutes the largest and to-date most reliable phylogenetic reconstruction of the family Chrysididae, which includes representatives of 186 species of cuckoo wasps. While the results of this phylogenetic reconstruction are consistent with previous ideas on the relationships of subfamilies and tribes, it shows the existence of several non-monophyletic genera (Chapter 3). CHC are involved in intraspecific recognition, often acting as contact sex pheromones. Nevertheless, it is not yet understood to what extent CHC profiles differ between the two sexes and whether some compound classes are more prevalent in one or the other sex. So far, no comparison of CHC profiles of males and females has been done for more than a dozen of related species. In Chapter 4, I describe and compare CHC profiles of females and males of 58 species of cuckoo wasps in order to evaluate whether and to what extent CHC profiles of these species differ between the sexes. I demonstrated that CHC profiles of cuckoo wasps are frequently (more than 90% of the species analyzed) and strongly dimorphic (both sexes of a given species tend to produce very different CHC compounds). Methyl-branched compounds tend to be more prevalent in males (especially dimethyl-branched compounds) and unsaturated compounds prevail in females. Moreover, a sex-specific pattern in the distribution of the double bond position of alkenes was evident: internal double bond positions (> 11) occur predominantly in males, whereas alkenes with the doublé bond at position 9 were more abundant and frequent in females (Chapter4). In Chapter5, I investigated how CHC profiles of cuckoo wasps differ across species. Are CHC profiles of cuckoo wasps species-specific, enabling their use as cues for species recognition? How do CHC profiles resemble phylogenetic relatedness? In Chapter 5, I try to answer these questions by comparing CHC profiles of 59 species of cuckoo wasps. CHC profiles of cuckoo wasps are shown to be species (and sex-) specific. I show that CHC profiles are useful as a complementary tool to help delimiting taxonomically difficult sibling species. Moreover, the evaluation of CHC profiles of five commonly occurring species within a genus, showed little or no geographical variation. However, CHC profiles of closely related species may differ strongly among each other, not being useful to track the evolutionary history of species (Chapter 5). Sexual selection is generally credited for generating striking sexual dimorphism by causing changes in male traits. Most often, sexual selection has a stronger effect on males, who compete for access to and may be selected by females, thus male traits may rapidly evolve. Nevertheless, in cuckoo wasps, it appears that it is the female sex the one evolving faster changes, with females of very closely related species showing extremely divergent profiles. One plausible reason for this disparity is that natural selection acting on female’s CHC profiles may be stronger than sexual selection on males (Chapter 6). Since females of cuckoo wasps are most probably engaged in an evolutionary arms race with their female hosts, CHC profiles of female cuckoo wasps are likely rapidly evolving, thus explaining part of the strong observed sexual dimorphism of CHC (Chapter 6). In fact, Chapter 7 shows evidence of a possible ongoing evolutionary arms race between five cuckoo wasps of the genus Hedychrum and their hosts. Hedychrum species parasitize either Coleoptera-hunting or Hymenoptera-hunting digger wasps. Since the coleopteran prey of the former digger wasps is naturally better protected against fungus infestation, these wasps do not embalm their prey with alkene-enriched secretions as do the Hymenoptera-hunting digger wasps. Thus, Coleoptera-hunting digger wasps can apparently diversify their profiles to escape chemical mimicry. Interestingly, only female cuckoo wasps of these hosts have started producing the same compound classes and even the same CHC compounds as those of their hosts. Male cuckoo wasps, however retain an alkene-enriched CHC profile that reflects the molecular phylogeny of the genus (Chapter 7). Whereas, a larger number of parasite-host comparisons may be needed to further conclude that an arms race between cuckoo wasps and their hosts is capable of generating sexual dimorphism of cuckoo wasps, this thesis constitutes the first effort towards this, providing a starting point for further studies. Finally, I provide some methodological tools that may help in speeding up the sometimes cumbersome process of analyzing and identifying CHC profiles. One of the most time-demanding steps in the processing of CHC data is the alignment of CHC chromatograms. This process is often done manually, because alignment programs are mostly designed for metabolomics or are just recently being developed. I analyzed CHC profiles using a combined approach with two freely available programs. I used AMDIS (Automated Mass Spectral Deconvolution and Identification System, http://chemdata.nist.gov/mass-spc/amdis/) to deconvolute and automatically identify all CHC of interest present in a chromatogram. I then developed a series of R scripts to correct for potential, unavoidable errors while processing CHC chromatograms with AMDIS. Chapter 8 explains this procedure. In the next chapter, I developed a program that helps in the identification of one commonly occurring class of hydrocarbons. The limited number of linear alkanes (only one per carbon atom) and their characteristic diagnostic ion allows a rapid and unambigous identification of these substances. In opposition, unsaturated and methyl-branched compounds are more difficult to identify, as a result of the much larger diversity of existing compounds. To identify unsaturated compounds a derivatization is necessary to determine the position of the double bond. Methyl-branched alkanes, however can be identified from the original chromatogram if their diagnostic ions are known. Nonetheless, polymethyl-branched alkanes (e.g., compounds with two or more methyl groups along the chain) are often difficult to identify, because they may appear in mixes (e.g., 3,7 diMeC27 and 3,9 diMeC27), and tables containing the diagnostic ions are not easily available. Therefore, I developed a program that creates a table with all possiblemethyl-branched compounds containing up to 4 methyl groups, and that provides their diagnostic ions and a calculated retention index. This may allow a much faster identification of the methyl-branched compound a researcher is dealing with, without having to lose time in the tedious calculations by hand. The program is able to correctly identify, or at least, greatly reduce the number of possible options for the identification of an unknown methyl-branched compound. Thus, using this tool, most methyl-branched compounds can be readily identified (Chapter 9). This thesis ends with a general discussion (Chapter 10). Overall, this work provides a comprehensive overview of the diversity of cuticular hydrocarbons of cuckoo wasps. The analyses presented here shed light on the emergence and evolution of interspecific diversity and intraspecific sexual dimorphism of CHC profiles. In addition, two technical methods have been developed that could greatly facilitate the CHC analysis of insects
Kutikulare Kohlenwasserstoffe (engl. „cuticular hydrocarbons“, CHC) sind Substanzen, die wir in größeren Mengen auf der Körperoberfläche von Arthropoden finden. Diese Moleküle aus Kohlenstoff- und Wasserstoffatomen haben trotz ihrer einfachen Struktur entscheidende Funktionen bei Insekten: Ihre wasserabweisende Eigenschaften geben den Insekten die Möglichkeit, den Wasserhaushalt zu regulieren und Austrocknung zu vermeiden. Darüber hinaus ermöglicht die Vielfältigkeit der CHC ihre Verwendung als Signale für eine breite Palette von Kommunikations-und Erkennungsprozessen. Obwohl die Erforschung von CHC in den letzten zwei Jahrzehnten einen großen Einblick in die Funktionen bei Insekten ermöglicht hat, gibt es immer noch Verständnislücken bezüglich der Evolution und Diversifizierung von CHC (Kapitel1). In der vorliegenden Dissertation habe ich anhand verschiedener Arten der Wespen Familie Chrysididae die Diversifizierungsmuster von CHC erforscht. Die meisten der Goldwespenarten in dieser Studie sind spezialisierte Parasitoiden oder Kleptoparasiten von hauptsächlich solitären Hymenopteren. Wirte von anderen Goldwespen sind auch Phasmatodea und Lepidoptera. Goldwespen sind besonders interesante Modellorganismen, um die Evolution von CHC zu untersuchen. Denn sie haben auf ihrer Kutikula chemische Anpassungen an die chemischen Oberflächen ihrer Wirte entwickelt, um bei dem Wirt zu vermeiden, dass ihre eigenen chemischen Signale bei der Eiablage erkannt werden. Für einige Vertreter der Familie Chrysididae wurden chemische Unscheinbarkeit/Unsichtbarkeit („insignificance“) und chemische Mimikry beschrieben. Bei ersterem, handelt es sich um die Reduzierung der Gesamtmenge der CHC auf der Kutikula, bei letzterem um die Nachahmung des CHC Profils des Wirtes. Zudem, deuten unveröffentlichte Daten darauf hin, dass chemische Nachahmung unter den Chrysididae weit verbreitet ist (Kapitel 2). Eine zuverlässige phylogenetische Rekonstruktion der Chrysididae ist notwendig, um die Evolution eines Merkmales, wie z.B. die Ausbildung eines CHC-Profils, zu verfolgen. Daher stellt der erste Teil dieser Arbeit die größte und bis heute zuverlässigste phylogenetische Rekonstruktion der Familie Chrysididae dar, welche Vertreter von 186 Arten von Goldwespen umfasst. Die Ergebnisse dieser Phylogenie stehen in Übereinstimmung mit vorherigen Studien über die Beziehungen zwischen Subfamilien und Triben der Goldwespen. Die Phylogenie deutet jedoch auf die Existenz mehrerer nicht-monophyletischer Gattungen in Chrysididae hin (Kapitel 3). CHC sind an der innerartlichen Erkennung beteiligt und fungieren manchmal als Kontakt-Sex-Pheromonen. Es ist jedoch noch nicht klar, inwieweit die CHC-Profile zwischen den beiden Geschlechtern differieren und ob einige Verbindungsklassen in dem einen Geschlecht häufiger als in dem anderen vorkommen. Bislang gibt es lediglich einen Vergleich von CHC-Profilen zwischen Männchen und Weibchen für weniger alseinDutzendverwandterArten.In Kapitel 4 werden die CHC-Profile von Weibchen und Männchen von 58 Goldwespenarten beschrieben und verglichen, um zu beurteilen, ob und in welchem Ausmaß, sich die CHC-Profile dieser Arten zwischen den Geschlechtern unterscheiden. Ich konnte zeigen, dass CHC-Profile von Goldwespen stark sexuell dimorph sind (Männchen und Weibchen der gleichen Art neigen dazu, sehr unterschiedliche CHC-Verbindungen zu produzieren), und dass dieser Dimorphismus sehr häufig vorkommt (mehr als 90% der untersuchten Arten). Methylverzweigte Verbindungen (insbesondere dimethylverzweigte Verbindungen) waren tendenziel bei Männchen häufiger und bei Weibchen waren ungesättigte Verbindungen häufiger. Darüber hinaus war ein geschlechtsspezifisches Muster in der Verteilung der Doppelbindungsposition von Alkenen offensichtlich: interne Doppelbindungspositionen (>11) treten vorwiegend bei Männchen auf, während Alkene mit der Doppelbindung an Position 9 bei Weibchen häufiger vorkommen (Kapitel 4). Im darauf folgenden Kapitel meiner Arbeit, beschäftige ich mich mit der Frage wie unterschiedlich CHC-Profile von Goldwespen zwischen Arten sind. Sind CHC-Profile artspezifisch, wie es zu erwarten wäre, wenn sie zur Arterkennung dienen? Gibt es Ähnlichkeiten in Bezug auf die phylogenetische Verwandtschaft der Arten? In Kapitel 5, versuche ich diese Fragen zu beantworten, indem ich die CHC-Profile von 59 Goldwespenarten vergleiche. Ich zeige, dass CHC-Profile von Goldwespen art- (und geschlechts-) spezifisch sind, und dass CHC-Profile als ergänzendes Werkzeug zur Abgrenzung von taxonomisch schwierigen Geschwisterarten nützlich sind. Darüber hinaus zeigt die Beurteilung der CHC-ProfilevonfünfhäufigvorkommendeArteninnerhalbeinerGattungwenigoder keine geografische Variation, was bei der Abgrenzung der Arten hilft. Allerdings können CHC-Profile nah verwandter Arten sehr unterschiedlich sein. Somit sind sie kein geeignetes Merkmal um die Evolutionsgeschichte von Arten nachzuvollziehen (Kapitel 5). Im sich daran anschließenden Kapitel, geht es darum, zu verstehen warum CHCProfile der meisten Goldwespenarten so auffallend unterschiedliche CHC-Profile zwischen Geschlechtern aufweisen. Beider sexuellen Selektion wird in der Regel erwartet, dass siedurch Veränderungen männlicher Merkmale zu einem auffälligen Sexualdimorphismus führt. Meistens wirkt die sexuelle Selektion stärker auf die Männchen aus als auf die Weibchen, weil sie um die Weibchen konkurrieren und von den Weibchen ausgewählt werden müssen. Daher wird erwartet, dass männliche Merkmale schneller evolvieren. Dennoch scheint das weibliche Geschlecht bei Goldwespen das Geschlecht zu sein, das schneller evolviert, was sich z. B. dadurch äußert, dass Weibchen sehr nah verwandter Arten extrem divergierende Profile zeigen (Kapitel 6). Ein plausibler Grund für diese Verschiedenheit zwischen den Weibchen nah verwandter Arten ist, dass die natürliche Selektion, die auf die CHC-Profile von Weibchen wirkt, stärker sein kann als die sexuelle Selektion bei den Männchen (Kapitel 6). Da die Weibchen der Goldwespen höchstwahrscheinlich in einem evolutionären Wettrüsten mit ihren weiblichen Wirten stehen, ist es möglich dass die CHC-Profile von Weibchen schnell evolvieren und somit den stark beobachteten sexuellen Dimorphismus von CHC in Goldwespen erklären (Kapitel 6). In Kapitel 7, werden Hinweise auf ein mögliches fortwährendes Wettrüsten zwischen fünf Goldwespenarten der Gattung Hedychrum und ihren Wirten aufgezeigt. Arten dieser Gattung parasitieren entweder Grabwespen die Coleoptera oder Hymenoptera als Nahrung für ihre Nachkommen jagen. Da die Coleoptera-Beute natürlicherweise besser gegen Pilzbefall geschützt ist, balsamieren diese Wespen ihre Beute nicht mit durch Alkene angereicherte Sekrete ein, im Gegensatz zu der anderen Gruppe der Grabwespen, die Hymenopteren als Futter verwerten. Daher diversifizieren Coleoptera-jagende Grabwespen offenbar ihre Profile stärker,um der chemischen Mimikry ihrer Parasitoiden zu entkommen. Interessanterweise haben nur weibliche Goldwespen dieser Coleoptera-jagende Wirte begonnen, die gleichen Substanzklassen und sogar die gleichen CHC-Verbindungen wie die ihrer Wirte zu produzieren. Männliche Goldwespen behalten jedoch ein durch Alkene angereichertes CHC-Profil, das die molekulare Phylogenie der Gattung Hedychrum widerspiegelt. Um jedoch eindeutiger zu beweisen, dass ein Wettrüsten zwischen Goldwespen und ihren Wirten den Geschlechtsdimorphismus von Goldwespen hervorbringt, wäre eine größere Anzahl von Vergleichen zwischen Goldwespen und ihren Wirten nötig. Nichtsdestotrotz ist diese Arbeit ein erster Versuch, den Geschlechtsdimorphismus von CHC in Goldwespen zu erklären und ein Ausgangspunkt für weitere Studien. Abschließend stelle ich einige methodische Werkzeuge vor, die helfen können, den bisher umständlichen Prozess der Analyse und Identifizierung von CHC-Profilen zu beschleunigen. Einer der zeitaufwendigsten Schritte bei der Verarbeitung von CHC Daten ist die Alinierung von CHC-Chromatogrammen. Dieser Prozess wird oft manuell durchgeführt, da Alinierungsprogramme für die Metabolomik konzipiert sind oder gerade erst entwickelt werden. Meine CHC-Profile habe ich mit einem kombinierten Ansatz mit zwei frei verfügbaren Programmen analysiert. Ich benutzte AMDIS (Automated Mass Spectral Deconvolution and Identification System), um die CHC in einem Chromatogramm zu dekonvolutieren und automatisch zu identifizieren. Ich habe weiterhin eine Reihe von R-Skripten entwickelt, um mögliche unvermeidbare Fehler bei der Verarbeitung von CHC-Chromatogrammen mit AMDIS zu korrigieren. In Kapitel 8 wird dieses Verfahren erläutert. Im darauffolgenden Kapitel stelle ich ein Programm vor, das ich für eine erleichterte Identifizierung einer häufig vorkommenden Verbindungsklasse von CHC entwickelt habe. Die begrenzte Anzahl von linearen Alkanen (nur eines pro Kohlenstoffatom) und ihre charakteristischen diagnostischen Ionen erlauben die schnelle und eindeutige Identifizierung dieser Substanzen. Im Gegensatz dazu sind ungesättigte und methylverzweigte Verbindungen auf grund der viel größeren Vielfalt möglicher Verbindungen deutlich schwieriger zu identifizieren. Für die Identifizierung ungesättigter Verbindungen ist eine Derivatisierung notwendig, um die Position der Doppelbindung zu bestimmen. Methylverzweigte Alkane können jedoch theoretisch vom ursprünglichen Chromatogramm unterschieden werden, sofern die diagnostischen Ionen bekannt sind. Trotz alledem sind polymethylverzweigte Alkane (z.B. Verbindungen mit zwei oder mehr Methylgruppen entlang der Kette) oft schwer zu identifizieren, da sie in Mischungen (z. B. 3,7 diMeC27 und 3,9 diMeC27) auftreten können. Ihre diagnostische Ionen müssen entweder berechnet werden oder in Tabellen, die nicht leicht verfügbar sind, gesucht werden. Ich entwickelte daher ein kleines Programm, das eine Tabelle erstellt mit allen möglichen methylverzweigten Verbindungen mit bis zu 4 Methylgruppen sowie deren diagnostischen Ionen und einem berechneten Retentionsindex. Dies erlaubt eine viel schnellere Identifizierung der richtigen methylverzweigten Verbindung, ohne dass ein Wissenschaflter Zeit für die mühsamen Berechnungen von Hand verlieren muss. Das Programm ist in der Lage, die Anzahl möglicher Optionen einer unbekannten methylverzweigten Verbindung korrekt zu nennen oder zumindest die Auswahl stark einzugrenzen und damit die Identifikation der Substanz stark zu erleichtern. Es ist daher zu erwarten, dass mit diesem Werkzeug die meisten methylverzweigten Verbindungen leicht identifiziert werden können (Kapitel9). Ich schließe meiner Dissertation mit einer allgemeinen Diskussion (Kapitel 10). Die vorliegende Arbeit stellt einen umfangreichen Überblick der Diversität von kutikularen Kohlenwasserstoffen von Goldwespen dar. Dieser Einblick kann uns helfen, die Bedeutung von CHC-Profilen für Arthropoden im Allgemeinen besser zu verstehen. Konkret beleuchten die durchgeführten Analysen die Entstehung und Evolution von interspezifischer Diverstität bzw. Ähnlichkeiten von CHC-Profilen und intraspezifischen sexuellen Dimorphismus von CHC-Profilen. Darüber hinaus wurden technische Methoden entwickelt, die zukünftige Arbeiten zu CHC Analysen von verschiedenen Insekten stark erleichtern könnten

碩士
國立臺灣大學
昆蟲學研究所
92
英文摘要 Detection of Callosobruchus maculatus and C. chinensis infesting adzuki bean by near-infrared spectroscopy and cuticular hydrocarbon analysis by Chun-Yun Cheng Graduate Institute of Entomology, National Taiwan University ABSTRACT The number of batches of quarantine on the imported agricultural products have dramatically increased from 81,110 in 2000 to 106,253 in 2002 when Taiwan became a member of World Trade Organization. It is important to develop a quick detection technique that can be applied to the quarantine on various products from all over the world. In this study, near-infrared spectroscopic method was used to detect the existence of eggs, larvae and adults of Callosobruchus maculatus or C. chinensis which are the major pests of adzuki bean (Vigna angularis) during storage. Gas Chromatography/Mass Spectrometer (GC/MS) was also used to identify the age and sex of these two pests by analysis of cuticular hydrocarbons. The accuracy of detecting larvae of the weevils infesting adzuki beans was 96.3% by a near-infrared spectrometer. The accuracy of differentiating the adults infesting the bean was 97.5%. To discriminate the larvae or adults of C. maculatus from that of C. chinensis, the accuracy was 91.7 and 100%, respectively. The accuracy of determing the sex of C. maculatus mixed with bean was 90.6%. For C. maculatus, there is a significant correlation between estimated and real age of larvae (R2 = 0.9344, SD = 2.19 day) and also between estimated and real age of adults (R2 = 0.9978, SD = 0.25 day). The accuracy was 100% when applied to determine the sex of C. chinensis mixed with bean. For C. chinensis there is a significant correlation between estimated and real age of larvae (R2 = 0.9229, SD = 2.38 day) and also between estimated and real ages of adults (R2 = 0.9907, SD = 0.36 day). The analysis of carbohydrates on the insect cuticles via the GC/MS can differentiate Callosobruchus maculatus from C. chinensis and determine their sexes as well as the estimated age. Key words: Near-infrared, Callosobruchus maculatus, Callosobruchus chinensis, GC-MS, adzuki bean, detection

碩士
國立彰化師範大學
生物學系
104
Tropical fire ant (TFA), Solenopsis geminata is one of the invasive ants in Taiwan. S. geminata has polymorphism in worker sizes, caste and division of labor. In social insects, the cuticular hydrocarbons (CHCs) play an important role in chemical communication, including serve as nestmate and caste recognition. In this study, the cuticular hydrocarbons of S. geminata were identified by Gas chromatography-mass spectrometry (GC-MS) from different worker sizes (major, medium and minor), labor (nurses, foragers and dead workers), and life cycle (larvae and pupae). The extracts are analyzed by GC-MS to investigate the labor division and roles of worker by the proportion of CHCs. The GC-MS results include two major venom alkaloids corresponding to cis C11 and trans C11. In addition, there are six major compounds of cuticular hydrocarbons which are 5-, 4-Me C20, C21, 5-Me C21, 4-Me C21, 7, 6, 5-Me C22 and 5-Me C23. The results show that the proportion of CHC compositions of major, medium and minor works is different. The comparison of CHCs compositions of larvae and pupae from four fire ant colonies excavated from four locations in Taiwan are similar. The results reveal that medium, minor, nurse and forager contains similar the proportion of CHC compositions. Results of this study demonstrate that the difference in the proportions of CHC compositions in different worker sizes, caste and division of labor in S. geminata.

In Drosophila melanogaster, chemical cues in the form of cuticular hydrocarbons play an important role in reproductive behavior. The social and genetic processes that regulate their expression, however, are poorly understood. The social environment has been shown to influence hydrocarbon display. In this study, the effect of population density on the expression of hydrocarbons was evaluated. I demonstrate that the production of certain hydrocarbons depends on the population density in which the animal is reared. Individual hydrocarbons fluctuate in quantity independently from one another but the peaks during a light-dark cycle are static depending mostly on chain length. The regulation of fly hydrocarbons which are density-dependent is shown to be sexually dimorphic. The RNAi knockdown of various putative lipid metabolism genes was also used to study hydrocarbon expression. This study reveals that lipid metabolism genes which are not obvious mediators of HC synthesis influence cuticular hydrocarbon profiles.

abstract: Of all the signals and cues that orchestrate the activities of a social insect colony, the reproductives' fertility pheromones are perhaps the most fundamental. These pheromones regulate reproductive division of labor, a defining characteristic of eusociality. Despite their critical role, reproductive fertility pheromones are not evenly expressed across the development of a social insect colony and may even be absent in the earliest colony stages. In the ant Camponotus floridanus, queens of incipient colonies do not produce the cuticular hydrocarbons that serve as fertility and egg-marking signals in this species. My dissertation investigates the consequences of the dramatic change in the quantity of these pheromones that occurs as the colony grows. C. floridanus workers from large, established colonies use egg surface hydrocarbons to discriminate among eggs. Eggs with surface hydrocarbons typical of eggs laid by established queens are nurtured, whereas eggs lacking these signals (i.e., eggs laid by workers and incipient queens) are destroyed. I characterized how workers from incipient colonies responded to eggs lacking queen fertility hydrocarbons. I found that established-queen-laid eggs, incipient-queen-laid eggs, and worker-laid eggs were not destroyed by workers at this colony stage. Destruction of worker-laid eggs is a form of policing, and theoretical models predict that policing should be strongest in incipient colonies. Since there was no evidence of policing by egg-eating in incipient C. floridanus colonies, I searched for evidence of another policing mechanism at this colony stage. Finding none, I discuss reasons why policing behavior may not be expressed in incipient colonies. I then considered the mechanism that accounts for the change in workers' response to eggs. By manipulating ants' egg experience and testing their egg-policing decisions, I found that ants use a combination of learned and innate criteria to discriminate between targets of care and destruction. Finally, I investigated how the increasing strength of queen-fertility hydrocarbons affects nestmate recognition, which also relies on cuticular hydrocarbons. I found that queens with strong fertility hydrocarbons can be transferred between established colonies without aggression, but they cannot be introduced into incipient colonies. Queens from incipient colonies cannot be transferred into incipient or established colonies.
Dissertation/Thesis
Ph.D. Biology 2012

abstract: The repression of reproductive competition and the enforcement of altruism are key components to the success of animal societies. Eusocial insects are defined by having a reproductive division of labor, in which reproduction is relegated to one or few individuals while the rest of the group members maintain the colony and help raise offspring. However, workers have retained the ability to reproduce in most insect societies. In the social Hymenoptera, due to haplodiploidy, workers can lay unfertilized male destined eggs without mating. Potential conflict between workers and queens can arise over male production, and policing behaviors performed by nestmate workers and queens are a means of repressing worker reproduction. This work describes the means and results of the regulation of worker reproduction in the ant species Aphaenogaster cockerelli. Through manipulative laboratory studies on mature colonies, the lack of egg policing and the presence of physical policing by both workers and queens of this species are described. Through chemical analysis and artificial chemical treatments, the role of cuticular hydrocarbons as indicators of fertility status and the informational basis of policing in this species is demonstrated. An additional queen-specific chemical signal in the Dufour's gland is discovered to be used to direct nestmate aggression towards reproductive competitors. Finally, the level of actual worker-derived males in field colonies is measured. Together, these studies demonstrate the effectiveness of policing behaviors on the suppression of worker reproduction in a social insect species, and provide an example of how punishment and the threat of punishment is a powerful force in maintaining cooperative societies.
Dissertation/Thesis
Ph.D. Biology 2011

Conflicts over reproduction are common in animal societies and they are especially pronounced in groups of totipotent individuals. Workers in the drywood termite Cryptotermes secundus are totipotent. They can gain direct fitness via dispersal as winged sexual or inheritance of the natal nest as neotenic replacement reproductive. In this study we examined the actual conflict behaviour in C. secundus, possible mechanism of conflict-regulation, and factors influencing the conflict intensity. Cuticular hydrocarbons provided the information about nestmates which is required for mechanisms of conflict-resolution to work: they were (i) caste-specific, (ii) honest signal of fertility in neotenic queens, as they are reflecting the underlying JH titres mediated via caste-specific Neofem4 expression, and (iii) may also be informative enough to allow kin discrimination. Kin discrimination in C. secundus depended on social context and the individuals’ developmental potential. Sterile soldiers for example showed nepotistic grooming independent from the social context, but affected by wood resource, in contrast to workers that did not react to varying relatedness. Individuals showed distinct behavioural profiles before and during conflict, reflecting their reproductive potential: most prominent in the distinction were the dominance behaviour butting and proctodeal trophallaxis. The proposed role of trophallaxis as honest signal and inhibitory means in regulating reproductive development in C. secundus could be confirmed. Conflict intensity was greatly influenced by wood resource: workers were more likely to stay and fight for inheritance within nests that are resource rich, while when food gets limited dispersal conflict was most pronounced. Thus, individuals did continually assess the ecological (resources) and societal conditions (presence of reproductives, relatedness) and adjusted their developmental decisions in order to maximize own fitness. The developmental potential is linked to the moulting interval, as only individuals in the sensitive phase are able to react to a changing situation like orphaning. Thus the sensitive phase, besides honest signalling (via cuticular hydrocarbon profiles and trophallaxis) might be an further mechanism potentially regulating conflict in C. secundus, as it restricts the number of individuals capable of becoming neotenic in a ‘fair lottery’ process.

abstract: The most abundantly studied societies, with the exception of humans, are those of the eusocial insects, which include all ants. Eusocial insect societies are typically composed of many dozens to millions of individuals, referred to as nestmates, which require some form of communication to maintain colony cohesion and coordinate the activities within them. Nestmate recognition is the process of distinguishing between nestmates and non-nestmates, and embodies the first line of defense for social insect colonies. In ants, nestmate recognition is widely thought to occur through olfactory cues found on the exterior surfaces of individuals. These cues, called cuticular hydrocarbons (CHCs), comprise the overwhelming majority of ant nestmate profiles and help maintain colony identity. In this dissertation, I investigate how nestmate recognition is influenced by evolutionary, ontogenetic, and environmental factors. First, I contributed to the sequencing and description of three ant genomes including the red harvester ant, Pogonomyrmex barbatus, presented in detail here. Next, I studied how variation in nestmate cues may be shaped through evolution by comparatively studying a family of genes involved in fatty acid and hydrocarbon biosynthesis, i.e., the acyl-CoA desaturases, across seven ant species in comparison with other social and solitary insects. Then, I tested how genetic, developmental, and social factors influence CHC profile variation in P. barbatus, through a three-part study. (1) I conducted a descriptive, correlative study of desaturase gene expression and CHC variation in P. barbatus workers and queens; (2) I explored how larger-scale genetic variation in the P. barbatus species complex influences CHC variation across two genetically isolated lineages (J1/J2 genetic caste determining lineages); and (3) I experimentally examined how CHC development is influenced by an individual’s social environment. In the final part of my work, I resolved discrepancies between previous findings of nestmate recognition behavior in P. barbatus by studying how factors of territorial experience, i.e., spatiotemporal relationships, affect aggressive behaviors among red harvester ant colonies. Through this research, I was able to identify promising methodological approaches and candidate genes, which both broadens our understanding of P. barbatus nestmate recognition systems and supports future functional genetic studies of CHCs in ants.
Dissertation/Thesis
Doctoral Dissertation Biology 2016