Made available in DSpace on 2014-10-09T12:43:54Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:10:34Z (GMT). No. of bitstreams: 1 06784.pdf: 2785176 bytes, checksum: f2b021da60503c5d9617c8d3ec337b80 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2015-04-20T12:31:48Z (GMT). No. of bitstreams: 1 Marcio Rodrigo Alecio.pdf: 5329646 bytes, checksum: 74fcb177f7eeca1140502ad6646743e5 (MD5) Previous issue date: 2012-08-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The abundance, potential insecticide and evidence of synergism among the major constituents of timbo extracts (Derris and Deguelia) and the need to control of Cerotoma tingomarianus Bechyné (Coleoptera: Chrysomelidae) and Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) stimulated the realization of this study, which aimed to evaluate the biological effects of timbo extracts and its purified fractions compared to rotenone, as well as combined in different concentrations, on adults of C. tingomarianus and S. frugiperda larvae. Samples timbo roots were collected of plants in the Acre and Amazonas states, and obtained extracts by hot extraction of dry material method. The timbo extracts were grouped into six distinct groups and the bioassays were carried out by means of topical contact, contact by contaminated surface (filter-paper) and intake of contaminated diet in a completely randomized design. The mortality values of two insect species were submitted to Probit analysis to determine the LC50, LD50 and LT50. The other variables were subjected to variance analysis, when possible, using the Scott-Knott test (P> 0.05) for grouping treatment means. The intoxication by intake of contaminated diet/leaves is the most effective mean of contact for adults of C. tingomarianus and S. frugiperda larvae. The toxicity of timbo species extracts is variable and is related to the chemical composition of the extracts, as well as at form of exposure and the target insect species. Rotenone alone was not toxic to adult C. tingomarianus and showed low toxicity to S. frugiperda larvae, enhancing their biological effects on the insects/larvae when associated with the fractions containing timbo extracts, constituent A, constituent B and deguelin. The toxicity of timbo extracts for C. tingomarianus adult may be related to the combined action of several constituents. Extracts containing high levels of rotenone and deguelin are indicators of toxicity to S. frugiperda larvae. The association the constituent A with constituent B probably causes mortalities in the pre-pupal and pupal weight of S. frugiperda. The extract of timbo G1 and G3 showed the highest toxicity to insects and can be considered as the most promising for the development of biotechnology products for the control of C. tingomarianus adults and S. frugiperda larvae. / A abundância, o potencial inseticida e indícios de sinergismo entre os constituintes majoritários de extratos de timbó (Derris e Deguelia) e a necessidade de controle de Cerotoma tingomarianus Bechyné (Coleoptera: Chrysomelidae) e de Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) estimularam a realização desta pesquisa, que objetivou avaliar os efeitos biológicos de extratos de timbó e de suas frações comparados à rotenona purificada, isoladamente e combinados em diferentes concentrações, sobre adultos de C. tingomarianus e de lagartas de S. frugiperda. Foram coletadas amostras de raízes de plantas de timbó nos estados do Acre e do Amazonas e obtidos extratos a quente utilizando clorofórmio como solvente. Os extratos das plantas de timbó foram agrupados em seis grupos distintos e os bioensaios foram desenvolvidos pelas vias de intoxicação de contato tópico, de contato por superfície contaminada e por ingestão alimentar, com delineamento experimental inteiramente casualizado. Os valores de mortalidade das duas espécies de insetos foram submetidos à análise de Probit para determinação das CL50, DL50 e TL50. As demais variáveis foram submetidas à análise de variância, quando possível, utilizando-se o teste de Scott-Knott (P > 0,05) para agrupamento das médias dos tratamentos. A intoxicação por ingestão alimentar é a via de contato mais efetiva para adultos de C. tingomarianus e lagartas de S. frugiperda. A toxicidade dos timbós é variável e está relacionada à composição química dos extratos, a forma de exposição e a espécie de inseto alvo. A rotenona utilizada isoladamente não foi tóxica para adultos de C. tingomarianus e apresentou baixa toxicidade para lagartas de S. frugiperda, potencializando seus efeitos biológicos sobre as lagartas quando associada com as frações dos extratos de timbó contendo deguelina e os constituintes A e B. A toxicidade dos extratos de timbó para adultos de C. tingomarianus pode estar relacionada à atuação conjunta de diversos constituintes. Extratos com elevados teores de rotenona e deguelina são indicadores de toxicidade para lagartas de S. frugiperda. A associação do constituinte A com o constituinte B provavelmente provoca mortalidades na fase pré-pupa e o peso de pupa de S. frugiperda pode ser reduzido pela atuação conjunta de vários constituintes dos extratos de timbó. O constituinte A pode atuar como antagonista de mortalidade de lagartas, provocar deformações e afetar a viabilidade de adultos de S. frugiperda. Os extratos de timbó G1 e G3 apresentam a maior toxicidade para os insetos e podem ser considerados como os mais promissores para o desenvolvimento de produtos biotecnológicos para o controle de adultos de C. tingomarianus e de lagartas de S. frugiperda.

A cana-de-açúcar (Saccharum spp.) é uma importante cultura na produção de alimentos e energia. Várias espécies de insetos podem causar sérios prejuízos econômicos à cultura da cana-de-açúcar. A broca da cana-de-açúcar (Diatraea saccharalis) é a praga de maior relevância por estar amplamente distribuída nas regiões canavieiras. O silenciamento gênico por RNA de interferência (RNAi) se tornou uma técnica amplamente estudada e utilizada nos mais diversos aspectos da biologia. Uma de suas aplicações é no controle de insetos-praga como uma alternativa de alta eficiência, especificidade e reduzido impacto ambiental. A ingestão de moléculas de RNA dupla fita (dsRNA) com identidade a regiões de genes essenciais de insetos-praga pode resultar no silenciamento destes genes, levando a fenótipos deficientes. Neste contexto, o presente trabalho teve como objetivo buscar genes alvos para o silenciamento com potencial para impedir o desenvolvimento normal da D. saccharalis e estabelecer uma forma de entrega do dsRNA eficiente para o teste de genes, visando assim validar o uso da técnica para a espécie. Por meio da clonagem de regiões de genes ortólogos já utilizados como alvo de silencimento em outras espécies de insetos (V-ATPase A, Receptor de Ecdisona e Arginina Kinase), e de genes com função específica identificadas após a caracterização do transcritoma de D. saccharalis (Juvenile Hormone Epoxide Hydrolase, Neverland e Quitina Sintase) foram conduzidos ensaios de RNAi. Foram realizados ensaios de dose resposta para o gene V-ATPaseem lagartas neonatas, onde a concentração selecionada por causar melhor redução na expressão do gene alvo foi de 2,5 µg µL-1. Esta concentração foi então utilizada em ensaios de alimentação para os outros genes. Os genes V-ATPase A, receptor de Ecdisona, Arginina Kinase, Juvenile Hormone Epoxide Hydrolase e Quitina Sintase apresentaram redução significativa no número de transcritos em larvas, demonstrando a viabilidade do uso de RNAi em D. saccharalisneonatas. O gene Neverland não demonstrou redução no acúmulo de transcritos nas condições trabalhadas. O gene GFP inicialmente utilizado como controle negativo apresentou variação na expressão de genes alvo, sendo desconsiderado como bom controle para D. saccharalis. O silenciamento dos genes alvo requer quantidades elevadas de dsRNA, superiores aos obtidos por transcrição in vitro, o que limita a viabilidade de ensaios com maiores replicatas e para determinar efeitos biológico. Alternativas de produção de dsRNA devem ser avaliadas para viabilizar a seleção de genes alvo efetivos / Sugarcane (Saccharum spp.) is an important crop for the production of food and bioenergy. Many insect species can cause economic losses in sugarcane. The sugarcane borer (Diatraea saccharalis) is the most important sugarcane pest, because it occurs in all production regions. Gene silencing by RNA interference (RNAi) rapidly became a widely investigated approach, adopted in various aspects of biology. One of the potential applications of RNAi is agricultural pest control, as an alternative with high efficiency, specificity and reduced environmental impact. The ingestion of double-stranded RNA (dsRNA) molecules with identity to regions of essential genes of the insect-pest can result in the target gene knock-down and, consequently, to deficient phenotypes. In the present work, target genes with the potential to affect the normal development of D. saccharaliswere searched, together with an efficient dsRNA delivery approach to test the target-genes to validate the use of the RNAi in D. saccharalis. Based on degenerated primers, expressed orthologous genes previously tested in other insect species (V-ATPase A, Ecdisone Receptor, and Arginine Kinase) were cloned,whilegenes with specific function (Juvenile Hormone Epoxide Hydrolase, Neverland, and Chitin Synthase) were identified from an in-house assembled transcriptome of D. saccharalis and cloned. A dose-response assay was conducted using the V-ATPase gene region delivered by droplets to neonate larvae, and the 2.5 µg µL-1dsRNA concentration was selected for further tests. This concentration was then used to deliver the other genes. The dsRNA version from the genes V-ATPase A, Ecdisone Receptor, Arginine Kinase, Juvenile Hormone Epoxide Hydrolase and Chitin Synthaseexhibited a significant reduction in the accumulation of transcripts, indicating the viability of RNAi to D. saccharalis in 1st instar larvae. The Neverland gene was not silenced by RNAi in the used conditions. The dsRNA of the Green Fluorescent Protein gene, used as negative control appeared to affectother gene targets. Target gene silencing require large amounts of dsRNA, more than what is achievable by in vitro transcription, which limits the viability to conduct large assays with more replicates and to determine biological effects. Alternatives to produce dsRNA need to be evaluated to enable the selection of effective target genes

The life-history and infection parameters of the entomopathogenic nematodes Steinernema feltiae (Filipjev)(Nematoda:Rhabditida) and Heterorhahditis megidis (Poinar, Jackson & Klein)(Nematoda:Rhabditida) were examined to provide specific details for the construction of mathematical SI models for biological control of soil insect pests. Laboratory experiments using the Greater Waxmoth, Galleria mellonella as the model host were undertaken to specifically examine the transmission behaviour of infective juvenile nematodes. The proportion of infective juveniles of S. feltiae which infected hosts was dependent on time. Previous studies declared that the proportion of infective juveniles which can infect is static, however, over a period of 5 days most of the infective juveniles infected hosts, demonstrating that the proportion infecting is dynamic. Infection of hosts by both species of nematode was compared using two mathematical representations of the transmission rate. Whereas the most parsimonious form of transmission for H. megidis was the linear Mass Action function, it was evident that, when measured at the individual nematode scale, S. feltiae transmission was non-linear. I postulated that this functional difference is due to the biology of the two species of nematodes. The subsequent effect of including the non-linear response on model predictions were investigated and it was demonstrated that the dynamics of the host nematode interaction became less stable. Spatial models of S. feltiae infection were parameterised from laboratory experiments, and control prediction of these models examined. The horizontal rate of dispersal through sand columns was determined in the presence and absence of hosts. Infective juveniles were found to disperse preferentially towards hosts. The predicted dynamics of pest control using the spatial moqel were highly dependent on the degree of nematode dispersal, host dispersal and the attraction of nematode infective juveniles towards hosts. The overall findings of this thesis have been placed in the context of epidemiological models created elsewhere, and predict that entomopathogenic nematodes may be targeted to specific pest systems with a high degree of success. An understanding of the infection biology of these nematode species is crucial in determining how and when pests may be controlled, and equally importantly, which systems successful control is not predicted.

Maize contributes substantially to food security in the Eastern Cape province of South Africa. It is a staple food to many of the province’s rural and urban inhabitants. Insect pests are one of the factors that hamper its productivity and as a result, deprive farmers of good yields. The adverse effects of insecticides and the high cost associated with them and the cost of transgenic seeds are some of the challenges faced by small-scale farmers in rural areas. Alternative control methods which include the use of indigenous techniques to control pests are now sought. A study to identify and document ethnobiological means used by rural farmers to manage insect pests of maize was conducted in the rural areas of the Eastern Cape Province. A total of 217 participants were interviewed on the matter, using semi-structured but detailed questionnaires. Rural farmers due to their linkage to agriculture activities and the fact that they are considered as custodians of agricultural indigenous knowledge were selected as respondents. Only maize producing and IsiXhosa speaking people were chosen to contribute. Main focus was on the demography of respondents, crop production activities and insect pest control. Pretesting of the questionnaire in order to assess the appropriateness of questions and comprehension by both farmers and enumerators was done. Data was analysed using descriptive statistics. Fifty five percent (55 %) of the respondents were females and the highest number of participants was from the Chris Hani District Municipality. Majority of the people were unemployed or pensioners. Most had only attended primary school and the mean age was 59 years. Apart from maize, respondents were cultivating other crops such as cabbage, Swiss chard, potatoes etc. Stalk borers followed by cutworms were the main pests of maize in these areas. Respondents used mainly insecticides, followed by alternative substances, which also included cultural control methods such as planting date manipulation. Few respondents used plants as control agents for insect pests. Some people did not control pests even though they were a problem in their fields. The most used plant was Chenopodium ambrosiodes L, while the most used substance was Madubula (a detergent). The most used insecticide was carbaryl from the carbamite family. Respondents listed different preparation techniques for all the control methods mentioned. These techniques revealed different times of preparation, quantities of ingredients, amounts applied on plants, modes of application and intervals of application. Rural farmers in the study areas used different atypical methods which may play a significant role in pest management today. Some of the products may have a positive influence on agriculture, while some are dangerous to humans and environmental health. Further research which will investigate their potential use in pest control needs to be done.

The common maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), a well known pest of stored-maize grain in most parts of the world, was identified as one of the major constraints of harvested maize grains in the Eastern Cape Province, South Africa. The use of plants or their products is one of the recent methods being investigated for insect pest control worldwide. Therefore, the main aim of the present study was to identify available plants in the Eastern Cape that could be used to combat the problem of Sitophilus zeamais in stored-maize grains. For the realization of the aims of this research, the following studies were carried out; a preliminary survey was conducted to obtain baseline information on the farmers’ knowledge and experience of indigenous insect pest control methods in the Eastern Cape. Also, studies on the insecticidal potential of the essential oils of some plants were investigated against the maize weevil. The quality parameters of maize grains treated with the essential oils was also studied and, using a rat model, the toxicity of the essential oils was investigated. The outcome from this study revealed that there is awareness amongst the farmers in the Eastern Cape on the use of plants or their products to control insect pests. Unfortunately, such methods are currently being neglected and the knowledge of their application was found to be eroding. Among the various essential oils screened were those from Mentha longifolia L. and Tagetes minuta L. which evoked an appreciable level of contact, fumigant and repellent toxicity on the maize weevil. Further work done to determine the effects of the oils on maize stored over a period of three months revealed that the two oils had no adverse effect on the proximate compositions and some quality parameters of the stored maize. However, the toxicological study conducted on rats showed that the oils at tested concentrations exhibited some level of toxicity. It is, therefore, suggested that the essential oils of M. longifolia and T. minuta should not be used to treat maize grains intended for human consumption.

M.Sc. (Zoology) / This study centres around the findings on mites (Acari: Eriophyidae) (also referred to as "eriophyids") as potential biological control agents of weeds in South Africa. The use of mites in the biological control of weeds is briefly reviewed. Four species of the family Eriophyidae have been used against weeds in other countries. Seventeen eriophyid species, causing symptoms possibly of significance to biological weed control of 12 plant species regarded as weeds in South Africa, are discussed. Methods to rear and study eriophyids in the laboratory have not yet been fully developed and studying these practically invisible arthropods, very closely associated with their host plants, frequently presents difficulties. Reported methods, and those used for the present study, are reviewed and discussed. The eriophyid, Aceria lantanae (Cook), causing flower and leaf galls on Lantana camara L. could not be successfully established in a quarantine laboratory on potted plants propagated from cuttings of L. camara forms from South Africa. The results obtained during this attempt were inconclusive. The failure of establishment on local L. camara forms could be attributed to a variety of factors, of which rearing methods and host incompatibility are the most likely. Convolvulus arvensis is a troublesome agricultural weed and occurs in especially the Orange Free State and the eastern and southern parts of the Cape Province. Biological control may possibly contribute to curbing this weed in South Africa. The eriophyid, Aceria malherbae Nuzzaci, causing deformation and galling on C. arvensis is regarded as one of the most promising candidates for the biological control of this weed. It was imported and successfully established on potted plants in a quarantine laboratory on biotypes of the weed occurring in South Africa. The biology and host specificity of A malherbae are accounted for, by using reported information and data obtained during this study. The biology of A. malherbae broadly conforms to the biology of other eriophyid species. This species has a narrow host range, being restricted to species very closely related to C. arvensis. The symptoms caused by this eriophyid on C. arvensis are described, and seem especially harmful to the reproductive potential of the plant. Two eriophyid species from L. camara from Paraguay, Tegonotus stefneseri and Paraphytoptus magdalenae, are described as new species. An illustrated as well as a dichotomous key to species (Eriophyoidea) described from L. camara, including the two new species, are provided. A key, and a review of known Tegonotus species, incorporated in a compendium based on the original descriptions of 74 species, are included. Shevtchenkella Bagdasarian is considered to be a junior subjective synonym of Tegonotus Nalepa.

RNA interference has enormous potential as a potent, specific, and environmentally friendly alternative to small molecule pesticides for crop protection. The use of exogenous double-stranded RNA offers flexibility in targeting and use in crops in which transgenic manipulation is not an option. The combination of RNAi with nanotechnology offers further advantages that are not available with dsRNA alone. In this work, I have evaluated several different combinations of nanomaterials and polymers for use in RNAi-based pest control systems. First, I have characterized the use of chitosan/dsRNA polyplex nanoparticles for gene knockdown using the model nematode Caenorhabditis elegans. Though chitosan/dsRNA polyplexes are equally as effective as naked dsRNA for gene knockdown on a concentration basis, these materials are assimilated into cells in a manner independent of dsRNA specific transport proteins. The mechanism of uptake is likely clathrin-mediated endocytosis. In addition, I identify a significant and yet unreported side-effect associated with chitosan exposure, the dysregulation of a major myosin isoform. Next, I have determined the efficacy of chitosan/dsRNA polyplex nanoparticles under different environmental conditions. The presence of inorganic ions (phosphate and nitrate) at realistic environmental concentrations does not alter the efficacy of the nanoparticles for gene knockdown, nor do they inhibit knockdown by naked dsRNA. These conditions did not cause any significant changes to the hydrodynamic diameter or zeta potential of the particles themselves between treatments. By contrast, a pH higher than six and the presence of natural organic matter significantly reduce the efficacy of the nanomaterials at gene knockdown but leave knockdown by naked dsRNA unaffected. Though some changes in polyplex size are observed in the pH treatments, these changes are comparatively small, and particles remain well within the size that can be ingested by C. elegans. At pH 8, the charge of the particles is effectively neutral. Similarly, concentrations of natural organic matter >2.5 mg/L cause a charge reversal of the particles, from strongly cationic to strongly anionic. Large aggregates are also visible in each of these treatments. Lastly, I characterize the efficacy of a suite of different polymer and solid core nanomaterials for dsRNA delivery, similar to the above. Poly-L-lysine, poly-L-arginine, Ge-doped imogolite, and poly-L-arginine-citrate coated Au nanoparticles all fail to cause any appreciable knockdown in the same C. elegans reporter system. Uptake of the polymers was exceedingly poor, and though the Au particles appear to have been ingested, there is no evidence of significant gene knockdown. Furthermore, poly-L-arginine caused significant injury to the mouthparts of C. elegans exposed to these materials. Layered double-hydroxide nanoparticles were successful at gene knockdown, and appear to function slightly better than naked dsRNA alone, and were translocated in C. elegans in a similar fashion to naked dsRNA. Taken together, these findings aid in the development of safe and effective RNAi biological control agents.

This research is a study of pest management systems in Malmö. Malmö is, as many other cities, growing and a node of people's movement. Occurrences of pests have increased in the last decade; this increase is attributed to travel as well as construction and building.What this paper shows is that the structures of pest management must evolve to be more communicative and collaborative. The thesis also adds to the housing crisis debate, as pests in one’s housing have a physical as well as psychological effect.

Thesis (MSc (Biomedical Sciences))--Cape Peninsula University of Technology, 2019 / Background: Baculoviruses have great potential as alternatives to conventional chemical insecticides. The large scale adoption of such agents has however been hampered by the slow killing times exhibited by these bio-insecticides, limitation to single target insect and difficulty of large scale production of these preparations. Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), initially identified in the Eastern Cape region of South Africa, has potential as a biocontrol agent as it possesses a higher speed of kill compared to other baculoviruses. Aims and methods: The main objective of this study was the identification, molecular characterisation and cloning of a structural core gene (polyhedrin) and three auxiliary genes, the inhibitor of apoptosis (iap2 and iap3) and the ecdysteroid UDP-glucosyltransferase (egt) genes, from TnSNPV in order to delineate its phylogenetic relationship to a Canadian isolate of the same virus and to other baculoviruses. In addition, the genes were expressed in an Escherichia coli (E. coli) based system as a prelude to genetic modification to increase the pesticidal property of the virus. Results: The genome size of the South African strain of TnSNPV was estimated at 160 kb and is significantly larger than the Canadian isolate of TnSNPV and may reflect genetic variation as the two strains have adapted to varying environmental conditions. Occlusion bodies of the South African strain of TnSNPV were visualised by Transmission Electron Microscopy and consisted of rod shaped single virions composed of a single enveloped nucleocapsid. Insect bioassays showed that the median lethal time (LT50) of the virus strain averaged 1.8 days which is significantly faster than other baculoviruses. The South African and Canadian strains of TnSNPV share nucleotide similarities greater than 95% for the genes analysed in this study, which indicates that they are closely related. From this analysis, the South African strain of TnSNPV identifies as a Group II NPV with the closest relatives being the Canadian strain of TnSNPV and ChchNPV. The topology of the tree for the polyhedrin protein was better resolved than that of the IAP2, IAP3 and EGT proteins and was comparable to the tree inferred from a concatenated data set consisting of complete polyhedrin/granulin, LEF8, and LEF9 proteins of 48 completely sequenced genomes. For the IAP2, IAP3 and EGT proteins, the separation of the lepidopteran and hymenopteran specific baculoviruses was not evident while the separation of Group I and II Alphabaculoviruses diverged from that observed from the baculovirus core gene polyhedrin as well as the tree inferred from complete polyhedrin/granulin, LEF8, and LEF9 proteins. Five distinct groups relating to IAP-1, 2, 3, 4 and 5 could be distinguished from the tree inferred from all IAP proteins from 48 fully sequenced baculoviruses. From this analysis, the IAP protein from the South African isolate of TnSNPV can be designated as an IAP3 due to sequence homology to other IAP3 proteins. Similarly, the IAP2 can be confirmed as an IAP2 protein as it clusters with other IAP2 proteins. RNA transcripts of the four genes were detected by RT-PCR at one hr after induction with Larabinose in BL21-A1 E. coli and persisted until four hrs post induction. Antisera directed against the C-terminal 6X His tag was able to detect the recombinant proteins at two hours after induction confirming the rapid rise in expression of the proteins which persisted at high levels until four hrs after induction. The discrepancy observed with the predicted molecular mass of the EGT protein and the migration on SDS-PAGE may be due to the absence of posttranslational modification in the E. coli expression system and the hydrophobic residues present in the N-terminal signal sequence. Conclusion: Sequence and phylogenetic analysis suggest that the two isolates of TnSNPV have been exposed to similar evolutionary pressures and evolved at similar rates and represent closely related but distinct variants of the same virus. The difference in genome size between the two strains is likely to reflect actual genetic differences as the strains have adapted to their local environments and hosts and the extent of the differences will only be apparent as more sequencing results become available. Phylogenetic analysis of the IAP and EGT proteins yields a tree that varies from the phylogenetic reconstruction observed for the polyhedrin gene as well as the concatenated data set consisting of complete polh/gran, LEF8, and LEF9 proteins and highlights the risks inherent in inferring phylogenetic relationships based on single gene sequences. The tree inferred from the concatenated data set of polh/gran, LEF8, and LEF9 proteins was a quick and reliable method of identification particularly, when whole genome data is unavailable and mirrors the accepted lineage of baculoviruses. Expression of the recombinant IAP2, IAP3, EGT and polyhedrin was confirmed by RT-PCR and immunoblot analysis and rose rapidly after induction and persisted at high levels. It is as yet unclear if the expressed proteins are functional particularly as post translation modifications are lacking in this system.