Arthropods are involved in the transmission of parasitic andviral agents that cause devastating diseases in animals andplants. Effective control strategies for many of these diseasesstill rely on the elimination or reduction of vector insect popu-lations. In addition to these pathogenic organisms, arthropodsare rich in microbes that are symbiotic in their associations andare often necessary for the fecundity and viability of their hosts.Because the viability of the host often depends on these obligatesymbionts, and because these organisms often live in closeproximity to disease-causing pathogens, they have been of in-terest to applied biologists as a potential means to geneticallymanipulate populations of pest species. As knowledge on thesesymbiotic associations accumulates from distantly related insect taxa, conserved mechanisms for their transmission and evolutionary histories are beginning to emerge. Here,Serap Aksoy summarizes current knowledge on the functionaland evolutionary biology of the multiple symbionts harbored inthe medically and agriculturally important insect group, tsetse,and their potential role in the control of trypanosomiasis.

Tsetse (Diptera: Glossinidae) are the vectors of Africantrypanosomes, the causative agents of sleeping sickness

disease in humans, as well as various diseases in animals.Recently, tsetse-transmitted diseases have been on therise, causing severe economic hardships in many alreadystressed communities. The extensive antigenic vari-ation the parasites display in their mammalian host hashampered efforts to develop effective vaccines, anddisease management strategies currently rely on thetreatment of infected hosts by chemotherapy and onreduction of tsetse challenge by eradication or sup-pression approaches. However, the success of activesurveillance attempts requires the development of im-proved methods with greater diagnostic sensitivitiesthan the traditional detection techniques1. The tryp-anocidal drugs used for treatment are expensive andhighly toxic, with adverse side effects; in addition,their efficacy has recently been challenged in the pres-ence of increasing parasite drug resistance detected inpatients2. Efforts to control vector populations havebeen based largely on ground and aerial spraying, aswell as the direct application of insecticides to cattle infarming communities. Recently, traps and targets havebeen used extensively to reduce vector challenge, butreports on their sustainability have been mixed be-cause their long-term success relies heavily on com-munity participation, awareness and resources. In geographically isolated areas, such as the island ofZanzibar, eradication attempts using the ‘sterile insecttechnique’ (SIT) have been successful, and have resulted

Tsetse – A Haven for MicroorganismsS. Aksoy

Serap Aksoy is at the Department of Epidemiology and PublicHealth, Section of Vector Biology, Yale University School ofMedicine, 60 College St., 606 LEPH, New Haven, CT 06510, USA.Tel: +1 203 737 2180, Fax: +1 203 785 4782, e-mail: serap.aksoy@yale.edu

19 Trees, A.J. et al. (1993) Prevalence of antibodies to Neospora caninum in a population of urban dogs in England. Vet. Rec. 132,125–126

20 Schares, G. et al. (1998) The efficiency of vertical transmission ofNeospora caninum in dairy cattle analysed by serological tech-niques. Vet. Parasitol. 80, 87–98

21 Osawa, T. et al. (1998) A multiple antigen ELISA to detectNeospora specific antibodies in bovine sera, bovine foetal fluids,ovine and caprine sera. Vet. Parasitol. 79, 19–34

22 Björkmann, C. et al. (1994) Neospora caninum in dogs: detection ofantibodies by ELISA using an iscom antigen. Parasite Immunol. 16,643–648

23 Björkmann, C. et al. (1997) An indirect enzyme linked im-munoassay (ELISA) for demonstration of antibodies of Neosporacaninum in serum and milk of cattle. Vet. Parasitol. 68, 251–260

24 Williams, D.J.L. et al. (1997) Novel ELISA for detection ofNeospora-specific antibodies in cattle. Vet. Rec. 140, 328–331

25 Packham, A.E. et al. (1998) A modified agglutination test forNeospora caninum – development, optimisation, and comparisonto the indirect fluorescent-antibody test and enzyme linkedimmunosorbent assay. Clin. Diagn. Lab. Immunol. 5, 467–473

26 Williams, D.J.L. et al. Evaluation of a commercial ELISA to detectserum antibody to Neospora caninum in cattle. Vet. Rec. (in press)

27 Paré, J. et al. (1995) Interpretation of an indirect fluorescent anti-body test for diagnosis of Neospora sp. infection in cattle. J. Vet.Diagn. Invest. 7, 273–275

28 Björkman, C. and Uggla, A. (1999) Serological diagnosis ofNeospora caninum infection. Int. J. Parasitol. 29, 1497–1507

29 Sasai, K. et al. (1998) A chicken anti-conoid monoclonal antibody identifies a common epitope which is present on motilestages of Eimeria, Neospora and Toxoplasma. J. Parasitol. 84, 654–656

30 Schares, G. et al. (1999) Serological differences in Neospora can-inum-associated epidemic and endemic abortions. J. Parasitol. 85,688–694

31 Wouda, W. et al. (1998) Serodiagnosis of neosporosis in individualcows and dairy herds, a comparative study of three enzyme-linkedimmunosorbent assays. Clin. Diagn. Lab. Immunol. 5, 711–716

32 Paré, J. et al. (1997) Neospora caninum antibodies in cows duringpregnancy as a predictor of congenital infection and abortion.J. Parasitol. 83, 82–87

33 Anderson, M.L. et al. (1997) Evidence of vertical transmission ofNeospora sp. infection in dairy cattle. J. Am. Vet. Med. Assoc. 210,1169–1172

34 Davison, H.C. et al. (1999) Herd-specific and age specific sero-prevalence of Neospora caninum in 14 British dairy herds. Vet. Rec.144, 547–550

35 Barber, J.S. and Trees, A.J. (1996) Clinical aspects of 27 cases ofneosporosis in dogs. Vet. Rec. 139, 439–443

36 Dubey, J.P. et al. (1997) Antibody responses of cows during anoutbreak of neosporosis evaluated by indirect fluorescent anti-body test and different enzyme-linked immunosorbent assays.J. Parasitol. 83, 1063–1069

37 Schares, G. et al. (1999) Bovine neosporosis: comparison of sero-logical methods using outbreak sera from a dairy herd in NewZealand. Int. J. Parasitol. 29, 1659–1667

38 Thurmond, M. and Hietala, S. (1995) Strategies to controlNeospora infection in cattle. Bovine Practitioner 29, 60–63

39 Cox, B.T. et al. (1998) Serology of a Neospora abortion outbreak ona dairy farm in New Zealand: a case study. N. Z. Vet. J. 46, 28–31

40 Barber, J.S. et al. (1997) Prevalence of antibodies to Neospora can-inum in different canid populations. J. Parasitol. 83, 1056–1058

41 Paré, J. et al. (1996) Congenital Neospora caninum infection in dairycattle and associated calfhood mortality. Can. J. Vet. Res. 60, 133–139

42 Björkmann, C. et al. (1996) Neospora species infection in a herd ofdairy cattle. J. Am. Vet. Med. Assoc. 208, 1441–1444

43 Harper, P.A.W. (1999) Are your cattle aborting? Agfact AO. 9. 58,1st edn 1994. New South Wales Agriculture

44 McAllister, M.M. (1999) Uncovering the biology and epidemiologyof Neospora caninum. Parasitol. Today 15, 216–217

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in complete elimination of disease from farming com-munities. On the mainland, however, this approach re-quires area-wide participation to be effective, as well asgeographically isolated populations to prevent reinva-sion of cleared lands. Because each individual method-ology has its limitations, there is growing interest in in-tegrated control programs that can incorporate severaldifferent schemes, depending on the ecology and epi-demiology of the disease, and on the cultural prefer-ences and resources of each community3. Given the re-cent advances in molecular technologies, there hasbeen an effort to develop alternative genetic-basedstrategies that can be included in the battery of toolsavailable for integrated control programs. As one suchapproach, transgenesis is being explored – it aims to modulate the vector competence of insects; ie. toeliminate the ability of insects to transmit diseaseagents by introducing and expressing foreign geneswith antipathogenic properties to interfere withpathogen viability, development and/or transmission.These pathogen-refractory insects can then replacetheir natural susceptible counterparts by virtue ofmechanisms that would confer reproductive advan-tages to the engineered insects4,5. Towards this end, ithas been possible to introduce foreign genes into severaldifferent insects by traditional transformation vectors(transposable elements or viral-transducing agents)6.In addition to this direct germ-line transformation ap-proach, foreign genes have also been expressed in thesymbiotic microorganisms that are closely associatedwith several arthropods, including tsetse4.

Characterization of tsetse symbiontsMany insects that have limited diets (such as blood,

plant sap or wood) throughout their entire develop-mental cycle rely on microbial symbionts for additionalnutrients not found in their restricted diet, and whichthey are unable to synthesize7,8. Microorganisms withdifferent ultrastructural characteristics have also beenreported from various tissues of tsetse, includingmidgut, hemolymph, fat body and ovaries9–13. Until re-cently, their taxonomic identification was unknown;however, PCR-based phylogenetic studies have nowshown that they represent three distinct organisms14.Two of these organisms are present in gut tissue: theprimary (P)-symbiont (Wigglesworthia glossinidia) re-sides intracellularly in the specialized epithelial cells(bacteriocytes), which form a U-shaped organ (bacteri-ome) in the anterior gut15,16, while the secondary (S)-symbiont (Sodalis glossinidius) is present in midgutcells17–19. The third organism, which has been charac-terized from reproductive tissue, is related to Wolbachiapipientis14. Tsetse are viviparous, retaining each eggwithin a uterus, where it hatches after fertilization; oneyoung larva matures and is finally expelled as a fullydeveloped third instar larva. Each female mates onceand can deposit 5–7 offspring during its 3–4 monthlifespan. During its intrauterine life, the larva receivesnutrients and both of the gut symbionts from its mothervia milk-gland secretions20,21. The Wolbachia bacteriumis present in the ovaries and is transovarially transmit-ted through maternal lineages. Given the unique repro-ductive biology of tsetse, however, all three symbiontsare, in essence, maternally acquired by the progeny.

It is difficult to study the individual functions of themultiple symbionts in tsetse. Attempts to eliminate the

symbionts by administration of antibiotics, lysozymeand specific antibodies result in retarded growth of theinsect and a decrease in egg production, preventing theability of the aposymbiotic host to reproduce22–24. Theability to reproduce, however, can be partially restoredwhen the aposymbiotic tsetse receive a bloodmeal thatis supplemented with B-complex vitamins (thiamine,pantothenic acid, pyridoxine, folic acid and biotin),suggesting that the endosymbionts play a role inmetabolism that involves these compounds25. In addi-tion to their role in nutrition, there has been indirect ev-idence suggesting that the presence of symbiont(s) mightalso enhance the establishment of trypanosome infec-tions in the midgut26. It has been shown that Sodalisproduces at least one type of chitinase enzyme, whichmight be responsible for increasing the trypanosomesusceptibility of its host insect27,28. It has been possibleto culture Sodalis in vitro29,30, and the availability of thisculture system has permitted the biochemical charac-terization of this organism. Carbon substrate assimilationtests suggest that Sodalis primarily utilizes N-acetyl-glucosamine and raffinose as its primary carbonsources in vitro19. The use of N-acetylglucosaminemight reflect an adaptation by the organism to the gutenvironment of its host, which is largely made of chitin(polymerized N-acetyl-D-glucosamine).

The W. pipientis-like symbiont detected in tsetse go-nads has been found to infect a wide range of inverte-brate hosts. In one survey in the neotropics, more than15% of the analyzed taxa were reported to carry thisgroup of microorganisms31. In the hosts they infect,Wolbachia have been shown to cause a variety of repro-ductive abnormalities, one of which is termed cyto-plasmic incompatibility (CI), and when expressed,often results in embryonic death owing to disruptionsin early fertilization events32. In an incompatible cross,the sperm enters the egg but does not successfully con-tribute its genetic material to the potential zygote. Inmost species, this results in very few hatching eggs.The infected females have a reproductive advantageover their uninfected counterparts as they can producesuccessful progeny with both the imprinted and nor-mal sperm. This reproductive advantage allows the in-fected insects to spread into populations. For func-tional studies, it has been possible to cure most insectsof their Wolbachia infections by administering antibi-otics in their diet; however, this approach has not beenfeasible in tsetse because the antibiotic treatment offlies results in the clearing of all bacterial symbionts,including the nutritional obligatory associations de-scribed above, and in the absence of these gut sym-bionts the flies become sterile. The analysis of tsetselaboratory colonies has shown that 100% of sampledindividuals carry Wolbachia infections, making it im-possible to investigate Wolbachia-mediated effects bytraditional mating experiments using infected and un-infected individuals. The prevalence of infections infield populations, however, has shown that varioustsetse species have been infected with Wolbachia andsignificant polymorphism exists in the field33. In onesurvey, although a 98% level of infection was seen inGlossina austeni sampled from South Africa, only 48%of the sampled Kenyan population was found to be infected33. Hence, Wolbachia-infected and -uninfectedlines can be developed from these heterogeneous fieldpopulations to elucidate the functional role of this

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organism in tsetse biology. A phylogenetic analysis ofthe Wolbachia strain types infecting different species oftsetse has shown that they are different and, as such,represent independent acquisitions33.

Evolutionary histories of symbionts Because it has been difficult to cultivate many of

these fastidious, and often intracellular organisms invitro, their correct taxonomic positioning has been con-troversial. Recent advances in PCR-based technologies,as well as the use of nucleic acid sequences in phylo-genetic reconstructions, have now provided additionalinsight into the relationships among bacteria34. The 16SrDNA sequence-based characterization of tsetse gutorganisms indicate that they are members of the fam-ily Enterobacteriaceae. The analysis of Wigglesworthiaand Sodalis from species representing the four subgeneraof Glossina: fusca, morsitans, palpalis and austeni, hasshown that they each form a distinct lineage in the g-subdivision of the Proteobacteria16 (Fig. 1). The phylo-genetic relationship of the different Glossina hostspecies has also been determined independently usingthe DNA sequence of the internal transcribed spacer 2

(ITS-2) regions of the rDNA locus35. The 16S rDNA se-quence of Wigglesworthia from different species oftsetse and the ITS-2 sequence of tsetse species havebeen found to be variable (5–10%) and, hence, wereused for phylogenetic reconstruction of the two histo-ries. When tsetse and Wigglesworthia phylogenies werecompared, they were found to display identical rela-tionships among the different species. This findingsuggests that a tsetse ancestor was infected with a bac-terium, and from this ancestral pair evolved the tsetsehost and endosymbiont, comprising the species oftsetse and Wigglesworthia strains that exist today andoverruling possible horizontal transfer events betweenspecies. The bacteriome-associated P-symbionts ofother insect taxa such as aphids36, whiteflies37, mealy-bugs38, cockroaches39 and carpenter ants40 have simi-larly been shown to represent distinct lineages thatparallel the evolutionary histories of their host insectspecies. Thus, although the microorganisms representdistant taxa in the Eubacteria, their evolutionary asso-ciations with their insect hosts are strikingly similar.The genome size of Buchnera, the P-symbiont of aphids,is 650-kb41, and the size of Wigglesworthia is also in this

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116 Parasitology Today, vol. 16, no. 3, 2000

Par

asito

logy

Tod

ay

(a) Glossina (b) BacteriaE. coli

G. fuscipes G. fuscipes -P

G. p. palpalis -P

G. tachinoides -P

G. p. gambiensis -P

G. m. morsitans -P

G. m. morsitans 2-P

G. m. centralis 1-P

G. m. centralis 2-P

G. austeni -P

G. brevipalpis -P

G. fuscipes -S

G. p. palpalis -S

G. m. morsitans -S

G. austeni -S

G. brevipalpis -S

S. zeamais -S

G. p. palpalispalpalis group

morsitans group

austeni group

fusca group

G. p. gambiensisG. tachinoides

G. pallipides-CG. pallipides-F

G. austeni-1G. austeni-2G. austeni-3G. austeni-4G. austeni-5G. austeni-6

G. brevipalpis-CG. brevipalpis-FG. longipennis

M. domesticaD. yakuba

G. m. morsitansG. m. submorsitansG. m. centralisG. swynnertoni

68 (81)

61

82

100

56

91

88

99

97

96

10014

2

88 (97)9

59 (100)28

8114

655100

39

10010

10027

999

Fig. 1. The phylogenetic placement of Glossina species and their primary (P)- and secondary (S)-symbionts.The phylogenetic tree constructed by maximum parsimony based on the internal transcribed spacer 2 (ITS-2) sequence of the indicated Glossina species (a). The analysis included 408 sites (432 with gaps), of which222 (246 with gaps) were variable and 88 (112 with gaps) were informative. One representative tree isshown (tree length 5 292; CI 5 0.897). The bootstrap confidence values (300 replications) are presentedat the nodes, the values in parentheses are bootstrap values where gaps have been included as weightedcharacters; numbers below denote branch length values. Drosophila yakuba and Musca domestica sequencesare used as outgroups. The grouping of the palpalis, morsitans, austeni and fusca species is indicated. F de-notes analysis done using field-collected material; C denotes colony material; numbers refer to indepen-dent PCR amplification, cloning and sequencing experiments. Phylogenetic analysis of Glossina symbiontsbased on their 16S rDNA sequence analysis (b). P denotes primary symbiont Wigglesworthia, and S denotesthe secondary symbionts analyzed from the corresponding species of Glossina. Escherichia coli was used asthe outgroup. The analysis included 893 sites of which 205 were variable and 121 were informative. Onerepresentative tree is shown (tree length 5 216; CI 5 0.843; bootstrap values for 500 replications areshown at the nodes). Abbreviations: CI, Consistency index; G. m. morsitans, Glossina morsitans morsitans; Glossina p. palpalis, G. palpalis palpalis. (Adapted with permission, from Ref. 35.)

range (S. Aksoy, unpublished), both much smallerthan their closely related, free-living relatives in theEnterobacteriaceae. The small sizes of their genomesmight be suggestive of their ancient intracellular histo-ries. Unlike the concordant relationship Wigglesworthiadisplays with its host, isolates of Sodalis from differenttsetse have almost identical 16S rDNA sequences, sug-gesting that they might either represent recent inde-pendent acquisitions by each host species, or alterna-tively, there may have been multiple horizontal transferevents between species (Fig. 1)21,33. In fact, comparativeanalysis of the 16S rDNA sequence of Sodalis with otherbacteria indicates a close relationship with the sym-bionts of Sitophilus zeamais and Acyrthosiphon pisum,indicating that this group of microorganisms might sharea recent ancestor within the Enterobacteriaceae.

Tissue tropism of symbiontsA PCR-based assay has been developed with sym-

biont-specific amplification primers to investigate thetissue tropism of the multiple symbionts in tsetse.Using this assay, Wigglesworthia is found exclusivelywithin the bacteriome-tissue, whereas Sodalis is detectedin midgut, muscle, fatbody, hemolymph, milkglandand in salivary glands of certain species18. Furthermore,the density of infections with Sodalis varies in the dif-ferent species analyzed. Whereas infections in G. morsitans and G. palpalis midgut tissues are maintainedat high density, infections in G. austeni and G. brevipalpisare significantly less dense, as measured by both PCR18

and microscopy42. The factors that control tissue trop-ism and the density of symbionts in insects are notknown. Although infections with Wolbachia were ini-tially thought to be largely restricted to the germ-linetissue of their insect hosts, they have recently beenshown to be present in a variety of somatic tissues inseveral different insects43,44. Analysis of the tissue trop-ism of Wolbachia in tsetse indicate that, whereas in G. morsitans and G. brevipalpis infections are restrictedto gonads, in G. austeni they can be detected in varioussomatic tissues, even in two-day-old teneral flies33. Theextensive infections associated with G. austeni somatictissues might reflect the particular phenotype of thebacterial strain harbored in this species, similar to thatreported in the Wolbachia popcorn strain characterizedfrom Drosophila melanogaster44. It remains to be seenwhether such an invasive tissue tropism in G. austenican also result in reduced fitness effects similar to thoseseen in D. melanogaster44.

Symbionts as expression vectorsThere is no direct germ-line DNA transformation

system available for tsetse and the viviparous nature ofits reproductive biology makes transgenic approachesthat rely on egg microinjections difficult. However, ithas been possible to culture the S-symbiont Sodalis invitro29,30, and a genetic transformation system has beendeveloped so that foreign genes can be introduced andexpressed in these cells30. The in vitro-manipulated re-combinant Sodalis can be acquired successfully by theintrauterine progeny by means of microinjection intothe female parent hemolymph. The recombinant sym-bionts are passed on to the F1 as well as their offspring,where they synthesize the marker gene product, greenfluorescent protein (GFP)18. Because Sodalis is found inclose proximity to the site of procyclic trypanosome

differentiation and replication in the midgut, the syn-thesis and secretion of antitrypanosomal gene productsin these symbionts in vivo can provide a mechanism todisrupt parasite establishment in the gut. However, forthe symbiont-based transformation approach to be suc-cessful, a significant proportion of the natural symbiontpopulation of the gut will need to be reconstituted byits recombinant counterpart so that foreign gene prod-ucts can accumulate in the gut to levels where they caninterfere with trypanosome biology. The eventual re-placement of parasite-susceptible vector populationswith engineered refractory flies could provide an addi-tional strategy to reduce disease in the field5. BecauseWolbachia infections in tsetse appear to express strongCI phenotypes, the two symbiotic systems could becoupled to drive the phenotypes conferred by the engi-neered gut symbionts into the field5. This should be fea-sible because Wolbachia infections rapidly invade popu-lations by virtue of the CI phenomenon they confer, andtherefore, they can drive other maternally inherited el-ements such as mitochondria45 or the engineered gutsymbionts into that same population46. Alternatively,the presence of Wolbachia infections in the somatic tis-sues of various insects, including tsetse, now opens upthe possibility of expressing antipathogenic genes di-rectly in this bacterium, which could then replace thesusceptible insect populations in the field33. Althoughno naturally occurring infectious transfer of Wolbachiahas been observed, it has become increasingly commonto transfer Wolbachia experimentally between differenthosts, and even into insects with no prior infection his-tory, making it an attractive gene expression systemwith a naturally associated driving mechanism47–50.

ProspectsUsing a similar symbiont-based insect transformation

approach, it has been possible to block the transmis-sion of Trypanosoma cruzi in Rhodnius prolixus in vivo byexpressing an anti-parasite peptide, cecropin A, in itssymbiont, Rhodococcus rhodnii in hindgut51. In the R. prolixus system, it has been possible to generate bugsfree from their natural gut symbiont flora and to intro-duce recombinant organisms into these aposymbioticfirst-instar animals through artificial coprophagy. Inaphids, the facultative S-symbiont (PASS), which isclosely related to Sodalis, has been introduced by mi-croinjection from Acyrthosiphon pisum into A. kondoiShinji (blue alfalfa aphid), as well as into A. pisum-nega-tive clones, where it is maintained in the progeny of theinjected mother aphids with a high rate of maternaltransmission52. The availability of in vitro symbiontculture and the relative ease of DNA transformationsystems in bacteria will facilitate the development ofsimilar disease intervention strategies in other medi-cally and agriculturally important vector systems suchas ticks, mites, bed bugs, lice, some species of fleas,planthoppers, white-flies and termites where these as-sociations have been firmly documented. The successof symbiont-based transgenic strategies in insects re-lies on a good understanding of the molecular and de-velopmental biology of the symbionts, as well as thepathogens transmitted by each system, so that geneswith transmission-blocking activities can be identifiedand expressed efficiently in the correct tissues, therebyadversely affecting pathogen viability. In addition toquestions regarding technical success and efficacy, there

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are many questions about the safety and regulatoryconcerns for release of genetically modified insects, es-pecially human-biting vectors. Before any release stud-ies can be entertained with recombinant animals, in-formation on environmental and ecological hazardsassociated with the releases and potential public healthrisks will need to be deliberated. The successful re-placement of vector populations with engineered in-sects will also depend on the availability of a soundknowledge base of the natural ecology and populationbiology of these insect species.

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