Ek Nguu

Determination of Heavy Metal Content in Water, Sediment and Microalgae from Lake Victoria, East Africa

Lake Victoria, which is the largest fresh water lake in Africa, represents a unique ecosystem that has the largest fresh water fishery in the continent. However, increased anthropogenic activities has increased the potential pollution of the lake especially the heavy metal pollutants which may be toxic to humans and aquatic fauna. There is need therefore for continuous monitoring of pollution levels in the lake. Samples of water, soil sediments and algae were collected in dry, long and short rainy periods of 2008 and analyzed for heavy metal by Atomic Absorption Spectrophotometry. The highest concentration of trace metals were found in sediment samples with Zn having the highest mean concentration values in both Winam (1.019 ppm) and Mwanza gulf (0.889 ppm). The mean concentration of Pb was higher in water samples from Winam gulf (0.823 ppm), while Hg in microalgae samples from Winam gulf had a mean concentration of 0.000148 ppm. The highest concentration of Zn (1.589 ppm) was determined in the sediment samples from Kirumba bay of the Mwanza gulf and the lowest was in sediments from Kishimba bay (0.327 ppm). Levels of trace metals in microalgae were not significant in different sites of the Mwanza Gulf. Like in the Mwanza gulf, levels of Zn was high in sediments from all the sites sampled in Winam Gulf, the highest recorded at Kisat. Pb levels were highest in the water samples from Hippo point, whereas concentration levels of Cd, Cr and Hg were lowest in all the four sites sampled. The maximum biomass of micro- algae occurred at Kisat during the short rain season (November-December) followed by Kamito in the same season.

Unlocking the potential of tropical root crop biotechnology in east Africa by establishing a genetic transformation platform for local farmer-preferred cassava cultivars

Cassava genetic transformation capacity is still mostly restricted to advanced laboratories in the USA, Europe and China; and its implementation and maintenance in African laboratories has remained scarce. The impact of transgenic technologies for genetic improvement of cassava will depend largely on the transfer of such capabilities to researchers in Africa, where cassava has an important socioeconomic niche. A major constraint to the development of genetic transformation technologies for cassava improvement has been the lack of an efficient and robust transformation and regeneration system. Despite the success achieved in genetic modification of few cassava cultivars, including the model cultivar 60444, transgenic cassava production remains difficult for farmer-preferred cultivars. In this study, a protocol for cultivar 60444 developed at ETH Zurich was successfully implemented and optimized to establish transformation of farmer-preferred cassava cultivars popular in east Africa. The conditions for production and proliferation of friable embryogenic calli (FEC) and Agrobacterium-mediated transformation were optimized for three east African farmer-preferred cultivars (Ebwanatereka, Kibandameno and Serere). Our results demonstrated transformation efficiencies of about 14–22 independent transgenic lines per 100 mg of FEC for farmer-preferred cultivars in comparison to 28 lines per 100 mg of the model cultivar 60444. The presence, integration and expression of the transgenes were confirmed by PCR, Southern blot analysis and histochemical GUS assay. This study reports the establishment of a cassava transformation platform at International Institute of Tropical Agriculture (IITA) hosted by Biosciences eastern and central Africa (BecA) hub in Kenya and provides the basis for transferring important traits such as virus resistance and prolonged shelf-life to farmer-preferred cultivars in east Africa. We anticipate that such platform will also be instrumental to transfer technologies to national agricultural research systems (NARS) in sub-Saharan Africa.

Linkage and mapping analysis of a non-susceptibility gene to densovirus (nsd-2) in the silkworm, Bombyx mori.

Nonsusceptibility to Bombyx mori densovirus type 2 (BmDNV‐2) is controlled by a recessive non‐susceptibility gene, nsd‐2 (non‐susceptibility to DNV‐2) in B. mori. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 (BF1) progeny were used for linkage analysis and mapping of nsd‐2 using silkworm strains C124 and 902, which are classified as being highly susceptible and non‐susceptible to DNV‐2, respectively. BF1 larvae were inoculated twice with DNV‐2 virus at the first and second instar stages. DNA was extracted from each of the surviving fifth instar larvae and analysed by RFLP inheritance patterns using probes specific to each of the 28 linkage groups of B. mori. Our results indicated that the non‐susceptibility gene was linked to linkage group 17, since all surviving larvae showed the homozygous profile of strain 902 in their genotype. The other linkage groups showed mixtures of heterozygous and homozygous genotypes, indicating an independent assortment. A linkage map of 30.6 cM was constructed for linkage group 17 with nsd‐2 mapped at 24.5 cM and three closely linked cDNA markers were identified.

Lipophorin from the tsetse fly, Glossina morsitans morsitans.

1. Lipophorin was isolated from the haemolymph of adult tsetse fly, Glossina morsitans morsitans, by ultracentrifugation in a potassium bromide density gradient. 2. The tsetse fly lipophorin (Mr congruent to 600,000) has a density of congruent to 1.11 g/ml and consists of two apoproteins, apolipophorin-I (apoLp-I, Mr congruent to 250,000) and apolipophorin-II (apoLp-II, Mr congruent to 80,000), both of which are glycosylated as shown by staining with periodate-Schiff reagent. The protein complex is composed of 49% protein and 51% lipids. 3. The finding of lipophorin in tsetse fly haemolymph suggests that, although these flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements.

Isolation and Properties of 600‐kDa and 23‐kDa Haemolymph Proteins from the Tsetse Fly, Glossina morsitans: their Possible Role as Biological Insecticides

The haemolymph of the tsetse fly. Glossina morsitans morsilans. contains a high (lipophorin) and a low molecular weight protein of high densities. I. 11 and 1.29 g/ml. respectively. The purification of the proteins was achieved by a combination of density gradient ultracentrifugation and reported gel permeation chromatography. The lipophorin is of high molecular weight (Mr∼ 600.000) and consists of two apoproteins. apolipophorin I (M Mr∼250.250,000) and apolipo‐phorin II (M Mr∼ 80.000) both of which are glycosylated. Lipophorin also has a pi of 6.1. However, electrophoresis under non‐denaturing and denaturing conditions showed the low molecular weight protein to be a single polypeptide chain (Mr∼ 23,000). Amino acid analysis revealed a relatively high content of the acidic amino acids as well as serine and glycine. The protein contained lipids as shown by Sudan Black staining but was unglycosylated. Using rabbit antiserum against the isolated protein in immunodiffusion and immunoblotting experiments, no cross‐reactivity was detected with haemolymph samples from insects representing six orders. In conclusion, the finding of lipophorin suggests that, although flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements. However, the results for the low molecular weight protein indicate that the protein is unique to Glossina, suggesting that it may have an important role in the physiology of this insect and is therefore a significant target for vector management.

Effects of heavy metal pollution on ω3 polyunsaturated fatty acids levels in tilapia fish from Winam Gulf of Lake Victoria

Winam Gulf is facing major pollution threats from anthropogenic input of pollutants such as heavy metals and agrochemical residues. This has deleterious effects on flora and fauna in the lake and consequently the quality of omega - 3 polyunsaturated fatty acids (PUFAs) which have numerous health benefits in humans. In addition, heavy metal bioac- cumulation in fish poses a threat to human health. The major objective of the study was to establish whether there is a cor- relation between the heavy metal pollutants and the levels of omega - 3 PUFAs in fish. Levels of heavy metals - lead, cadmium, Zinc and chromium in sediments, water, and tilapia from selected sites in Winam Gulf were investigated. They were analyzed using Atomic Absorption Spectrophotometer. Fish muscles were further analyzed for omega -3 PUFAs us- ing gas chromatography. Sediment samples accumulated the highest levels of heavy metals ranging from below detection limit to as high as 277 mg/kg on dry weight basis. Zinc levels in fish muscles were the highest whereas cadmium was the lowest. Heavy metal levels in water were found to be lowest compared to sediments and fish. Omega-3 PUFAs, particu- larly alpha-linolenic, eicosapentaenoic, docosapentaenoic and docosahexaenoic acids were in substantial amounts in fish. Oil contents were in the range of (2.47- 3.87) %. There was no clear link observed between the levels of heavy metals and omega-3 PUFAs in fish although the fish muscles showed presence of these metals.

Dominant allele phylogeny and constitutive subgenome haplotype inference in bananas using mitochondrial and nuclear markers

Abstract Cultivated bananas (Musa spp.) have undergone domestication patterns involving crosses of wild progenitors followed by long periods of clonal propagation. Majority of cultivated bananas are polyploids with different constitutive subgenomes and knowledge on phylogenies to their progenitors at the species and subspecies levels is essential. Here, the mitochondrial (NAD1) and nuclear (CENH3) markers were used to phylogenetically position cultivated banana genotypes to diploid progenitors. The CENH3 nuclear marker was used to identify a minimum representative haplotype number in polyploids and diploid bananas based on single nucleotide polymorphisms. The mitochondrial marker NAD1 was observed to be ideal in differentiating bananas of different genomic constitutions based on size of amplicons as well as sequence. The genotypes phylogenetically segregated based on the dominant genome; AAB genotypes grouped with AA and AAA, and the ABB together with BB. Both markers differentiated banana sections, but could not differentiate subspecies within the A genomic group. On the basis of CENH3 marker, a total of 13 haplotypes (five in both diploid and triploid, three in diploids, and rest unique to triploids) were identified from the genotypes tested. The presence of haplotypes, which were common in diploids and triploids, stipulate possibility of a shared ancestry in the genotypes involved in this study. Furthermore, the presence of multiple haplotypes in some diploid bananas indicates their being heterozygous. The haplotypes identified in this study are of importance because they can be used to check the level of homozygozity in breeding lines as well as to track segregation in progenies.

Characterization of protective antigens from the midgut of Amblyomma variegatum ticks

Separation of midgut membrane proteins from the tick,Ambylomma variegatum, using a non-ionic detergent (TritonX-114), resulted in two protein fractions, namely DET (detergent) and AQ (aqueous). In immunoblotting analysis with polyclonal antibodies against these fractions, 4 proteins (Mr ∼27,000, 67,000, 86,000 and 95,000,)and 2 proteins (Mr ∼54,000 and 67,000) were detected in the DET and AQ fractions, respectively. Three of the DET fraction proteins Mr∼27,000, 67,000 and 95,000 were glycosylated since they bound to the lectin, concanavalin A. In 2-dimensional gel electrophoresis, the AQ and DET fraction proteins were found to be acidic in nature. In a series of bioassay experiments, rabbits were first immunised with both DET and AQ fractions and then infested with ticks. The egg batch weights of these ticks were reduced by 50% compared to control ticks. Furthermore, there was a significant reduction in the hatchability of eggs laid by ticks fed on rabbits previously immunised with both DET (14%) and AQ (33%) fractions. Based on the egg hatchability, there productive capacity of ticks was reduced by 77 and 48% by DET and AQ fractions, respectively.

Expressed Centromere Specific Histone 3 (CENH3) Variants in Cultivated Triploid and Wild Diploid Bananas (Musa spp.)

Centromeres are specified by a centromere specific histone 3 (CENH3) protein, which exists in a complex environment, interacting with conserved proteins and rapidly evolving satellite DNA sequences. The interactions may become more challenging if multiple CENH3 versions are introduced into the zygote as this can affect post-zygotic mitosis and ultimately sexual reproduction. Here, we characterize CENH3 variant transcripts expressed in cultivated triploid and wild diploid progenitor bananas. We describe both splice- and allelic-[Single Nucleotide Polymorphisms (SNP)] variants and their effects on the predicted secondary structures of protein. Expressed CENH3 transcripts from six banana genotypes were characterized and clustered into three groups (MusaCENH-1A, MusaCENH-1B, and MusaCENH-2) based on similarity. The CENH3 groups differed with SNPs as well as presence of indels resulting from retained and/or skipped exons. The CENH3 transcripts from different banana genotypes were spliced in either 7/6, 5/4 or 6/5 exons/introns. The 7/6 and the 5/4 exon/intron structures were found in both diploids and triploids, however, 7/6 was most predominant. The 6/5 exon/introns structure was a result of failure of the 7/6 to splice correctly. The various transcripts obtained were predicted to encode highly variable N-terminal tails and a relatively conserved C-terminal histone fold domain (HFD). The SNPs were predicted in some cases to affect the secondary structure of protein by lengthening or shorting the affected domains. Sequencing of banana CENH3 transcripts predicts SNP variations that affect amino acid sequences and alternatively spliced transcripts. Most of these changes affect the N-terminal tail of CENH3.

Properties of a Factor in Eland Plasma that Inhibits Transformation of Bloodstream-form Trypanosoma brucei brucei

Fresh plasma from the African eland, Taurotragus oryx, contains a factor that inhibits the transformation of bloodstream-form Trypanosma brucei brucei into procyclic (midgut) forms. Heating of the plasma at 42, 50 and 60 °C for 30 min resulted in a 20, 38 and 40% loss of inhibitor activity respectively, whereas only negligible loss occurred below 42 °C. Similarly, one and four freeze-thawing cycles resulted in 32 and 60% loss of activity respectively. Inactivation of the inhibitor activity, which occurred rapidly during storage (for example, 80% loss after 7 days at −20 °C) could not be stopped by the addition of various protease inhibitors or lyophilisation of the plasma. Treatment of plasma with pronase (1 mg/ml for 2 h) completely abrogated the inhibitor activity, whereas trypsinisation had only a partial effect. Ammonium sulphate fractionation of fresh plasma showed that the inhibitor was insoluble above 50% salt. When the plasma was fractionated by anion-exchange chromatography, the inhibitory activity was recovered in the bound fractions. Efforts to purify the inhibitor were unsuccessful due to the rapid loss of activity under all conditions tested. It is concluded that the low capacity of eland blood to support transformation of bloodstream trypanosomes is due to an inhibitor present in the plasma fraction.RésuméLe plasma frais de l’élan africain, Taurotragus oryx, contient un facteur qui inhibe la transformation des formes sanguines de Trypanosoma brucei brucei en formes procycliques (intestinales). Le chauffage du plasma à 42, 50 et 60 °C pendant 30 min provoque 20, 38 et 40% de perte de l’activité inhibitrice respectivement, alors que la perte est négligeable en dessous de 42 °C. De même, un et quatre cycles de congélation-décongélation provoquent respectivement 32 et 60% de perte d’activité. L’inactivation de l’activité inhibitrice, qui intervient rapidement pendant le stockage (par exemple, 80% de perte après 7 jours à −20 °C) ne peut pas être arrêtée par l’addition de différents inhibiteurs de protéases ou la lyophilisation du plasma. Le traitement du plasma avec de la pronase (lmg/ml pendant 2 heures) supprime complètement l’activité inhibitrice, alors que la trypsinisation a seulement une effet partiel. Le fractionnement par le sulphate d’ammonium du plasma frais indique que l’inhibiteur est insoluble au dessus de 50% de sel. Quand le plasma est fractionné en Chromatographie par échange d’anions, l’activité inhibitrice est retrouvée dans les fractions liées. Les tentatives de purification de l’inhibiteur ont échoué à cause de la perte rapide d’activité pour toutes les conditions testées. On en conclut que la faible capacité du sang d’élan à favoriser la transformation des formes sanguines du trypanosome est dû à un inhibiteur présent dans la fraction plasmique.