Journal of Insect Physiology 54 (2008) 17–24 Eicosanoids mediate melanotic nodulation reactions to viral infection in larvae of the parasitic wasp, Pimpla turionellae Yonca Durmus-a, Ender Bu¨yu¨kgu¨zela, Burcin Terzia, Hasan Tunazb, David Stanleyc,Ã, Kemal Bu¨yu¨kgu¨zela aDepartment of Biology, Faculty of Arts and Science, Karaelmas University, Zonguldak, Turkey bDepartment of Plant Protection, Faculty of Agriculture, Kahramanmaras- Su¨tc-u¨ I˙mam University, Kahramanmaras-, Turkey cUSDA/Agricultural Research Service, Biological Control of Insects Research Laboratory, 1503 S. Providence Rd., Columbia, MO 65203, USA Received 1 February 2007; received in revised form 21 March 2007; accepted 21 March 2007 Nodulation is the quantitatively predominant insect cellular immune function activated in response to bacterial, fungal and some viral infections. We posed the hypothesis that parasitoid insects express melanotic nodulation reactions to viral challenge and that eicosanoidsmediate these reactions. Treating fifth-instar larvae of the ichneumonid endoparasitoid Pimpla turionellae with Bovine Herpes SimplexVirus-1 (BHSV-1) induced nodulation reactions in a challenge dose-dependent manner. Experimental larvae treated with thecyclooxygenase inhibitor, indomethacin, the lipoxygenase inhibitor, esculetin, and the phospholipase A2 inhibitor, dexamethasone,resulted in severely impaired nodulation reactions to our standard BHSV-1 challenge dose. The immunoinhibitory influence ofdexamethasone was reversed in larvae reared on culture medium amended with arachidonic acid, the fatty acid precursor of eicosanoidbiosynthesis. Larvae that had been reared on media amended with indomethacin, esculetin, or dexamethasone were also compromised intheir nodulation reactions to viral challenge. The influence of the orally administered pharmaceutical was expressed in a dose-dependentmanner. Finally, wasp larvae reared in the presence of indomethacin and dexamethasone expressed significantly decreased levels ofphenoloxidase activity in response to viral challenge. These findings draw attention to the idea that endoparasitoid insects express cellularimmune reactions to viral challenge; they also support our hypothesis that eicosanoids mediate nodulation reactions to viral challenge inthese highly specialized insects.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Parasitoid wasp; Pımpla turıonellae; Cellular immunity; Nodulation; Phenoloxidase; Eicosanoids biological actions, eicosanoids act in several aspects ofinsect immunity. Two categories of insect innate immune Eicosanoids are produced by enzymatic oxygenation of reactions are appreciated. Humoral reactions involve arachidonic acid (AA) and two other C20 polyunsaturated induced biosynthesis of various anti-microbial peptides fatty acids. The two major groups of eicosanoids are prostaglandins and the many lipoxygenase products.
nodulation, encapsulation and phagocytosis ( Virtually all animals are thought to biosynthesize a wide range of eicosanoids, which serve in a large (but unknown) number of molecular, physiological and ecological actions bacterial infection are mediated by eicosanoids. This eicosanoid hypothesis has been supported by reports, onover 20 insect species, from several laboratories ( ). Looking in more detail than nodulation, Corresponding author. Tel.: +1 875 573 5361x245; eicosanoids also mediate particular steps that lead to 0022-1910/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
Y. Durmus- et al. / Journal of Insect Physiology 54 (2008) 17–24 cell spreading, a distinct phase of nodulation, and phagocytosis are mediated by eicosanoids in larval waxmoths, Galleria mellonella. found that The cyclooxygenase (COX) inhibitor, indomethacin eicosanoids mediate hemocyte elongation in hemocytes (1-(p-chlorobenzoyl)-5-methoxy-2-methyl-3-indolyl-acetic acid), isolated from tobacco hornworms, Manduca sexta. Eico- the 5- and 12-lipoxygenase (LOX) inhibitor esculetin (6,7- sanoids also mediate microaggregation reactions (another dihydroxycoumarin), the PLA2 inhibitor dexamethasone step in nodulation) to bacterial challenge ( [(11b,16a)-9-fluoro-11-17,21-trihydroxy-16-methylpregna- 1,4-diene-3,20-dione], arachidonic acid (AA), dopamine, bovine serum albumin (BSA), and Folin-Ciolcalteu Re- reported that eicosanoids mediate behavioral fever re- agent were purchased from Sigma-Aldrich (St. Louis, MO, sponses to infection in the locust Schistocerca gregaria. The eicosanoids also act in insect humoral immunity. found that biosynthesis of antibacterial proteins also depends on eicosanoids in the silkworm,Bombyx mori. And reported a Before injection, larvae were anesthetized by chilling functional coupling between the immune deficiency path- them on ice for 5 min and surface-sterilized by swabbing way and eicosanoid biosynthesis in Drosophila. We surmise their cuticles with 95% ethanol (EtOH). Injections were eicosanoids are crucial elements in the mediation of in carried out with a 10 ml Hamilton micro-syringe (Hamilton, Reno, NV). Injections were performed dorso-laterally in With respect to the nature of infecting organisms, the abdominal segments with the pharmaceutical treat- eicosanoids act in insect defense reactions to several species ments and BHSV-1 challenge doses on opposite sides. The of bacteria, two fungal species and eggs of a parasitoid abdomen was palpated gently after injection to mix the contents of the hemocoel. Eicosanoid biosynthesis inhibi- tors (EBIs), indomethacin, esculetin and dexamethasone mediate melanotic nodulation reactions to Bovine Herpes Simplex Virus-1 (BHSV-1) challenge in larvae of the Control insects were injected with 70% EtOH (5 ml) and greater wax moth, G. mellonella. At a higher level of experimental wasps with EBIs (5 mg/5 ml EtOH). All larvae biological organization, parasitoid insects can become were immediately challenged with BHSV-1 in a standard exposed to viral infection via interactions with their hosts.
dosage of 2 Â 104 PFU in 5 ml of culture medium, except in We posed the hypothesis that parasitoids also express dose–response experiments, following the injection proto- melanotic nodulation reactions to viral challenge and that eicosanoids mediate these reactions. In this paper, we at selected times after injections. The larvae were anesthe- report on experiments with the ichneumonid endoparasi- tized by chilling them on ice, then their hemocoels were toid Pimpla turionellae designed to test our hypothesis.
exposed to count melanized, brownish-black nodules undera stereomicroscope at 45 Â . After initial counting, thealimentary canals were removed and nodules in theremaining internal tissues were then counted. The nodules were distinct and direct counting reliably reflected theextent of the nodulation response to infections A colony of P. turionellae was maintained in the laboratory at 2371 1C, 7575% RH, and a photoperiodof 16-h light:8-h dark. Wasp larvae were reared on the Several control experiments were conducted to deter- pupae of the greater wax moth, G. mellonella. Adults were mine the level of background nodulation in the wasp fed daily with a 50% filtered honey solution and G.
larvae. Because the larvae were reared in non-sterile mellonella pupal hemolymph every other day. Adult wasps conditions, control experiments (no treatment) were were allowed to oviposit in wax moth pupae. Fifth-instar performed to register the background number of nodules P. turionellae (20–30 mg) larvae were used in the nodula- in the larvae. Nodulation in unchallenged larvae was tion reaction and PO activity experiments.
assessed by randomly taking 10 fifth-instar larvae from Stock BHSV-1 was kindly donated by Dr. Aykut O¨zkul artificial diet, chilling them on ice, and counting the (Veterinary Faculty, Ankara University). The virus stock nodules, as described above. The effect of wounding on was kept at À70 1C until used. The concentrations of virus nodulation was determined by wounding 10 larvae with the solutions (4 Â 106, 4 Â 105, 4 Â 104 plaque-forming units needle of the micro-syringe. Nodulation was assessed 1 h (PFU)/ml) were prepared by serially diluting the original post-injection (PI). The effect on nodulation of the COX liquid suspension with distilled water. Dilutions of the inhibitor indomethacin, the LOX inhibitor esculetin, and stock solution were made immediately before injection.
the phospholipase A2 inhibitor dexamethasone, BHSV-1 Y. Durmus- et al. / Journal of Insect Physiology 54 (2008) 17–24 and their solvents ethanol and distilled water, respectively, was tested in unchallenged larvae in the same concentra- Composition of chemically defined diet for rearing P. turionellae larvae tion (5 mg/larva for indomethacin and 2 Â 104 PFU/larva for virus) and volume (5 ml/larva for both solvent) as used for challenge larvae. Nodulation was assessed at 1 h PI.
2.5. Time-course of nodulation: influence of BHSV-1 Five groups of larvae (n ¼ 10) were chilled on ice and injected with 5 ml BHSV-1 (2 Â 104 PFU/larva) as de- scribed. The number of nodules was counted at 1, 2, 4, 6, For the dose–response curve, four groups of larvae ¼ 10) were chilled on ice and injected with 5 ml BHSV-1 2 Â 104 PFU per larva. Control larvae were injected with distilled water. Nodulation was assessed at 1 h PI as Eight groups of larvae (n ¼ 10) were tested. Individuals in the first group were injected with 5 ml ethanol (70%) as control. The second group was injected with 5 ml BHSV-1 (2 Â 104 PFU/larva). Groups 3–5 were injected with in- domethacin, esculetin, or dexamethasone (5 mg/larvae).
Groups 6–8 received similar EBI treatments, and were tion was assessed at 1 h PI as described above.
2.8. Oral treatments with EBIs and BHSV-1 Experimental wasp larvae were used for per os experi- ments to determine nodulation reaction and PO activity.
The synthetic diet described by was used for rearing first instar wasp larvae. The diet consisted of anamino acid mixture, a lipid mixture, an inorganic saltmixture, glucose, RNA and a mixture of water-soluble water to achieve the final concentrations of 0.001, 0.01 and vitamins The methods used in the preparation of the diets and dispensing these diets into test-tubes, To obtain wasp larvae, wax moth pupae were para- obtaining the eggs and first instar larvae and their sitized. The wasp eggs were recovered by dissecting the inoculation onto diets were described elsewhere parasitized hosts in saline solution (0.8% solution of NaCl). The larvae were allowed to hatch in the saline prepared in two parts. A vitamin–glucose mixture was prepared first. The remaining diet components were All the feeding experiments were started by inoculating prepared and autoclaved for 15 min at 121 1C for dissolving the newly hatched larvae into 10 mm  75 mm tubes the agar. Finally, the parts were combined in a sterile glove charged with 0.5 ml diet. Each tube received one larva and was closed with a rubber stopper. The tubes were kept Desired amounts of indomethacin, esculetin and dex- in the dark except during a short daily observation amethasone were first diluted in 1 ml of ethanol (70%) and period. Larvae that died during first 24 h and tubes that completed with distilled water to prepare solutions of the became contaminated during the assay period were required concentrations. Solutions of EBIs were then excluded from the experiments. A group of larvae were added into the diets before gel formation. The final reared on the diet without EBIs in each experiment as no- volumes of the diets were completed with double distilled treatment controls. The experiments were conducted under Y. Durmus- et al. / Journal of Insect Physiology 54 (2008) 17–24 the same laboratory conditions as mentioned for the The larvae fed the control diet and the diets amended Data were analyzed by one-way ANOVA. To determine with EBIs were inoculated with BHSV-1 (2  104 PFU/ significant differences between means least significant larva) by intrahemocoelic injection as described. At 1 h PI, difference (LSD) test () was used. When the nodulation was assessed. Each feeding experiment was F-estimate exceeded the probability of 0.05, the differences replicated four times with 10 larvae. Fifth-instar larvae were also collected for determination of phenoloxidaseactivity.
The outcomes of our control experiments are displayed The artificial diet for the rescue experiments contained in Untreated wasp larvae or larvae injected with 13.5 mg/100 ml AA instead of linolenic acid, the main the indicated substances yielded about 5–20 nodules/larva.
ingredient of dietary lipid mixture. Desired amounts of We note particularly that larvae challenged with EtOH or dexamethasone were prepared as just described.
the EBI esculetin yielded 16 and 20 nodules/larva, Four groups of fifth-instar wasp larvae (n ¼ 10) were respectively, while larvae exposed to all other treatments tested. One group of larvae was reared on control diet produced 10 or less nodules/larva. Larvae challenged with (insect artificial diet) as a negative control. Individuals in a BHSV-1 at 2 Â 104 PFU/larva produced over 50 nodules/ second group were reared on the diet amended with ethanol (EtOH, 70%) to control for the influence of EtOH.
A third group was reared on the diet amended with 100 mg 3.2. The influence of viral challenge dose on nodulation of dexamethasone. A fourth group of larvae was reared onthe diet amended with 100 mg of dexamethasone plus AA.
The data presented in show that nodulation The larvae in these groups were challenged by injection reactions to BHSV-1 challenge were expressed in a dose- with BHSV-1 (2 Â 104 PFU/larva) and immune parameters related manner in P. turionellae larvae. We recorded about were recorded at 1 h PI. Each experiment was replicated 15 nodules/larva in control wasps which increased to about four times with 10 larvae/replication.
55 nodules/larva in wasps treated with the highest dose of2 Â 104 PFU/larva. Each viral dose resulted in significantlyincreased nodulation and the increases obtained in a linear 2.10. Determination of phenoloxidase (PO) activity Larvae were surface sterilized in 95% ethanol. Ten intact fifth-instar larvae were homogenized at 4 1C by an 3.3. Time-course of nodulation: influence of BHSV-1 ultrasonic homogenizer (Bandelin Sonoplus, HD2070,Berlin, Germany) at 50 W, 40–50 s in 100 mM sodium We recorded the influence of incubation time after phosphate buffer (pH 7.0), and subsequently centrifuged at infection on nodule numbers (). Highest nodulation 10,000g for 10 min. Clear supernatant was used in PO (circa 55 nodules/larva) was recorded at 1 h PI. Longer activity assays. The samples were diluted 1:10 (v/v) incubation periods yielded significantly decreased numbers in 90 ml of sodium phosphate buffer. Phenoloxidase of nodules. We counted about 40 nodules/larva at 2, 4 and (EC1.14.18.1) activity was assayed by using a methodmodified from and as The results of background control experiments supernatant (50 ml) was added to 1.5 ml glass spectro-photometer cuvettes containing 1 ml phosphate buffer (100 mM, pH 7.0). After 20 min, 100 ml dopamine (10 mM in sodium phosphate buffer) was added and phenoloxidase activity (mOD/min) was determined by measuring absor- bance at 492 nm at 5-min intervals for 30 min at 30 a UV–vis spectrophotometer (Shimadzu 1700, Kyoto, Japan). Enzyme activity is expressed in absorbance unit (au)/min/mg protein at 492 nm. Assays were replicated four times each with 10 larvae. Protein concentrations in the supernatants were determined by the method of Fifth-instar larvae were treated as indicated and nodulation was assessed using bovine serum albumin as quantitative 1 h later as described in Section 2. Nodulation values are mean numbers of nodules (7S.E., n ¼ 10 larvae in all treatments).
Y. Durmus- et al. / Journal of Insect Physiology 54 (2008) 17–24 Number of Nodules
Number of Nodules
EtOH Virus
Virus Virus Virus
Virus concentrations (PFU/larva)
Fig. 1. The influence of BHSV-1 challenge dosage on nodulation reaction Fig. 3. Influence of eicosanoid biosynthesis inhibitors on BHSV-1 induced in P. turionellae larvae. Wasp larvae were injected with the indicated nodulation reactions in P. turionellae larvae. Larval Group 1 (EtOH) was dosage of BHSV-1 (distilled water for zero). Nodulation was assessed at injected with 5 ml of 70% ethanol as a negative control. Group 2 (Virus) 1 h PI as described in Section 2. Each point represents the mean number of was injected with our standard BHSV-1 challenge (2 Â 104 PFU/larva).
nodules (7S.E.; four replications (n ¼ 10 larvae)/treatment). Points Groups 3, 4 and 5 were injected with, respectively, indomethacin (Indo), annotated with the same letter are not significantly different (P40.05 esculetin (Esc) and dexamethasone (Dex), all at our standard dose of 5 mg/larva. Group 6 (Indo+virus) was treated with indomethacin prior toBHSV-1 injection. Group 7 (Esc+virus) was treated with esculetin priorto BHSV-1 injection. Group 8 (Dex+virus) was treated with dexametha- sone prior to BHSV-1 injection. The histogram bars represents the meannumber of nodules (7S.E.; four replications (n ¼ 10 larvae)/treatment).
Bars annotated with the same letter are not significantly different (P40.05 about 55 nodules/larva. Larvae treated BHSV-1 plus indomethacin, esculetin or dexamethasone yielded very low numbers of nodules, on par with background control 3.5. Arachidonic acid reversed the influence of displays the results of the AA rescue experiment.
Wasp larvae reared on the control diet and challenged with virus yielded the expected 50–55 nodules/larva. Larvaereared on diet amended with EtOH yielded about 40 Fig. 2. Time-course for nodulation reactions in response to BHVS-1challenge in P. turionellae larvae. Wasp larvae were injected with BHSV-1 nodules/larva, a significant reduction from control values.
(2 Â 104 PFU/larva). At the indicated times, PI nodulation was assessed as Insects reared on control diet supplemented with dexa- described in Section 2. Each point represents the mean number of nodules methasone produced very few nodules, approximately (7S.E.; four replications (n ¼ 10 larvae)/treatment). Points annotated 5–10/larva, in response to viral infection. Finally, wasp with the same letter are not significantly different (P40.05 (LSD test)).
larvae reared on the AA plus dexamethasone-supplemen-ted diet yielded about 50 nodules/larva, statistically similar 6 h PI and even fewer nodules at 8 h PI. Nodulation was assessed at 1 h PI in subsequent experiments.
3.6. The influence of per os administration of EBIs on 3.4. The influence of EBIs on nodulation reactions to viral Orally administered EBIs influenced wasp larval nodula- The influence of EBIs on nodulation is presented in tion and PO activity reactions to viral infection ( . We recorded about 10–20 nodules/larva in wasps Larvae reared on diets supplemented with dexamethasone, challenged with EtOH, indomethacin, esculetin or dex- indomethacin or esculetin, then challenged with viral amethasone. Larvae challenged with BHSV-1 yielded infection yielded fewer nodules than control larvae.
Y. Durmus- et al. / Journal of Insect Physiology 54 (2008) 17–24 maximal level very quickly, in about 1 h PI. Third, injection treatments with indomethacin, esculetin and dexametha- sone severely impaired nodulation reactions to viral challenge. Fourth, the hindering influence of dexametha-sone on nodulation was reversed in wasp larvae reared on media amended with AA, the biochemical precursor to eicosanoid biosynthesis. Fifth, per os administration of EBIs via culture media supplementation rendered experi- mental larvae severely weakened in nodulation and PO Number of Nodules
activity reactions to viral challenge. We interpret these findings with respect to the eicosanoid hypothesis, viz., eicosanoids are crucial mediators of insect cellular im- The work reported in this paper extends and strengthens Control diet
Dex+virus Dex+AA+virus
the idea that eicosanoids act in insect nodulation reactions to viral infection, recently put forth by . Although it is thought that eicosanoids act in Fig. 4. The influence of dietary AA on the inhibitory effect ofdexamethasone (Dex) on nodulation reactions of P. turionellae larvae.
hemocytic immunity of the social hymenopteran, Apis Larvae in Group 1 (control+virus) were reared on control diet and then mellifera (), this is the first report of injected with the standard BHSV-1 challenge dose. Group 2 larvae (EtOH) eicosanoid actions in the cellular immunity of a parasitic were reared on the diet amended with ethanol and then injected with hymenopteran. Because most research on parasitoids is BHSV-1. Group 3 larvae (Dex+virus) were reared on the diet amended focused on host immune reactions to parasitoid attack, this with Dex and then injected with BHSV-1. Group 4 larvae (Dex+AA+virus) of larvae were reared on the diet amended with Dex and AA and work draws attention to the idea that parasitoids also then injected with BHSV-1. Nodulation was recorded at 1 h PI. The experience, and defense themselves from, infections. This histogram bars represent the mean number of nodules (7S.E.; four idea broadens appreciation of host–parasitoid relations.
replications (n ¼ 10 larvae)/treatment). Bars annotated with the same This work also strengthens the earlier work by showing letter are not significantly different (P40.05 (LSD test)).
that the immunity-crippling effects of dexamethasone werereversed in wasp larvae reared on basal culture medium The influence of all three pharmaceutical compounds was supplemented with AA. AA-driven reversals of the expressed in a dose-related manner. For dexamethasone dexamethasone influence on cellular immunity have been and indomethacin, nodulation was reduced from about 50 nodules/larva in the absence of dietary EBI to less than 20 ever, this work shows that dietary administration of AA nodules/larva in the presence of the highest dose, 100 mg/ can also reverse the dexamethasone effect. Insects are able 100 ml diet. The results with esculetin differed slightly as to take up dietary AA, which in lengthy feeding experi- nodulation was reduced from a high of about 60 nodules/ ments can alter the overall fatty acid profile of tissue larva to 30 nodules/larva for larvae reared in the presence We recorded a similar pattern for the influence of EBIs Our data on the time-course of nodulation is interesting on PO activity For larvae reared in the presence in view of earlier studies of nodulation of indomethacin and dexamethasone, PO activity at 1 h PI was reduced in a dose-dependent manner. The results were pattern generally recorded shows increasing numbers of considerably different for larvae reared in the presence of nodules appear with increased incubation times PI, up to esculetin. In this experiment, PO activity was significantly about 4–6 h PI. The numbers of nodules did not further higher relative to control larvae reared in the absence of increase following longer incubation periods. In our studies with P. turionellae, we recorded about 55 nodules/larva at1 h PI, but the numbers of nodules decreased with longer incubation periods. Nodules are generally lashed tointernal organs or body walls, where they remain through In this paper, we report on the outcomes of experiments life. They are apparently cleared from the wasp larvae after designed to test the hypothesis that BHSV-1 challenge the initial virus-stimulated nodulation reaction. This may provokes eicosanoid-mediated nodulation reactions and be a general feature of cellular immunity in parasitoid stimulates eicosanoid-mediated increases in PO activity in wasps. The wasps may also be subject to bacterial and larvae of the ichneumonid wasp, Pimpla turionellae. Several fungal infection. We do not yet know whether the wasp points bolster the hypothesis. First, BHSV-1 challenge larvae form nodules in reaction to bacterial or fungal stimulated nodulation reactions that increased in a dose- challenge, nor how long bacteria- or funal-induced nodules related pattern with increasing viral challenge. Second, the would remain visible within the body cavities. This remains time-course experiment showed that nodulation reached a Y. Durmus- et al. / Journal of Insect Physiology 54 (2008) 17–24 Indomethacin
/mg protein/min)
Number of nodules/Larva
PO activity(au
Treatment (g/100 mL Diet)
Fig. 5. The influence of orally administered indomethacin, esculetin and dexamethasone on P. turionellae larval nodulation reactions (Panel a) and POactivity (Panel b) reactions to intrahemocoelic BHSV-1 challenge. Larvae were reared on media amended with the indicated EBI concentration. Fifth-instar larvae were challenged by BHSV-1 injection and immune parameters were recorded at 1 h PI. Each point represents the mean number of nodules(7S.E.; four replications (n ¼ 10 larvae)/treatment). Points annotated with the same letter are not significantly different (P40.05 (LSD test)).
Our selection of EBIs sheds light on the actions of We recently addressed the potential roles of eicosanoids eicosanoids in nodule formation. Larvae reared in the in PO activation and our rapidly growing body of presence of dexamethasone, indomethacin and esculetin knowledge on physiological defenses against viral challenge were impaired in their nodulation reaction to BHSV-1 challenge. Dexamethasone inhibits phospholipase A2, the commentary here. With respect to the possible roles of first step in eicosanoid biosynthesis. Indomethacin is a eicosanoids in viral replication in insect cells, we are now cyclooxygenase inhibitor and esculetin is a lipoxygenase working with a cell line established from Helicoverpa zea inhibitor. Because both EBIs curtailed nodulation reac- (HzAM1 cells). This cell line is non-permissive to tions, we infer that prostaglandins (cyclooxygenase pro- AcMNPV baculovirus replication. Our preliminary experi- ducts) as well as various lipoxygenase products act in ments indicate that treatments with EBIs somehow change nodulation reactions to viral challenge, as seen in many this non-permissive line into semi-permissive one. This insect species following bacterial challenge ( finding, along with other work using mosquito cells (cited ). The idea that several different eicosanoid species act in the nodulation process is consistent with limited to prostaglandins, act in anti-viral immune func- our understanding that nodulation is a complex process tions at the cellular and intracellular levels.
that results from a large number of individual cell actions.
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Our Synod Partner: The Right Reverend James Tengatenga Our synod partner this year is The Right Reverend James Tengatenga, from the Diocese of Southern Malawi. Bishop James has an impressive resume. He has studied in the U.S., England and Malawi, and was consecrated Bishop of Malawi in 1998. He has a Ph.D. in Church and State Relations from the University of Malawi, and has honorary degree

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