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Effects of cyclic nucleotides on midgut infections and maturation of T. b. brucei in G. m. morsitans

Cyclic nucleotide signalling through cyclic adenosine monophosphate (cAMP) is thought to play an important role in the transformation of the long slender (dividing) form to the short-stumpy (arrested) form in the mammalian bloodstream but the role of cyclic nucleotides in the tsetse-based part of the trypanosome life cycle is unknown. In a series of in vivo experiments, it was found that cyclic guanosine monophosphate (cGMP) but not cAMP could induce significantly higher rates of midgut infection in tsetse. Continuous feeding of either cGMP or cAMP to tsetse had no effect on rates of maturation of established midgut infections suggesting that these two parts of the life cycle in tsetse are not linked.


Findings
The short stumpy form of the trypanosome is thought to be pre-adapted to life in the tsetse fly, the long slender form maturing into the short stumpy form once a certain density of infection is reached in the mammalian host [1]. Similar processes happen in the tsetse fly with trypanosomes going through several transformations, starting in the midgut of the fly as bloodstream forms, they transform to procyclic forms before terminal differentiation into mammalian infective forms in the salivary glands (Trypanosoma brucei s.l.) or mouthparts (Trypanosoma congolense). A link between cAMP and cell cycle signalling in the trypanosome life-cycle was suggested by addition of cAMP analogues in vitro which promoted the long slender dividing stage to form non-dividing short stumpy forms [2].
In the present work Glossina morsitans morsitans were infected with T. b. brucei (stock Buteba 135, see MacLeod et al. [3]) on the day following the day of emergence from the puparium. Infective feeds were given in vitro using thawed stabilates of trypanosomes suspended in defibrinated ovine blood. To examine the effects of cAMP and cGMP on midgut infection establishment, flies received to a final concentration 1, 10 or 100 µM 8-Br-cGMP or 8-Br-cAMP (Sigma, UK) with their infective bloodmeal (cyclic nucleotides were dissolved in saline then added to the bloodmeal while control flies received saline only).
To examine the effect of 8-Br-cGMP post-infection 100 µM 8-Br-cGMP was added to the bloodmeal 48, 72, 96 or 120 h post infection. Flies which did not take the infective or supplemented feed were removed from the experiment.
To examine the effects of cyclic nucleotides on the maturation of trypanosomes, all flies received a bloodmeal containing 100 µM 8-Br-cGMP and were then either fed 100 µM 8-cAMP or 100 µM 8-Br-cGMP from the second feed onwards. Flies which did not take the infective feed were removed from the experiment.
Following infection, flies were maintained at 25°C and 70% relative humidity and fed on defibrinated ovine blood through an artificial membrane. To determine rates of establishment, flies were dissected 10 d post-infection (or 10 d post-treatment) and midguts were examined for the presence of trypanosomes by phase-contrast microscopy (X400). To determine rates of maturation, flies were dissected 28 d post-infection and midguts and salivary glands were examined for the presence of trypanosomes by phase-contrast microscopy (X400).
Generalised linear models with binomial errors were used to examine the proportion of flies with midgut infections (number of midgut infections/total number of flies dissected) or proportion of midgut infections maturing into mature infections (transmission of infectivity -TI: number of salivary gland infections/number of midgut infections) when compared to control flies (see MacLeod et al. [4]).
The effects of 8-Br-cGMP or 8-Br-cAMP on trypanosome midgut infection rates in male G. m. morsitans are shown in Figure 1. Addition of 8-Br-cGMP to the bloodmeal at concentrations of 10 or 100 µM significantly (p < 0.001) increased midgut infection rates from 16% control (number of flies dissected, n = 95) to 51% (n = 106) and 92% (n = 86) respectively. Addition of 1 µM 8-Br-cGMP resulted in midgut infection rates of 13% (n = 97) and was not significantly different (p = 0.817) from the control. There was no significant difference in infection rates between male and female flies fed the same concentrations of 8-Br-cGMP (data not shown).
In the present work, results show that cAMP does not appear to be involved in either the establishment of midgut infections or the maturation of established midgut infections in tsetse. This contrasts with in vitro work where cAMP signalling was shown to be involved in transformation of the replicating long-slender form to the cell  arrested short-stumpy [2]. However, we have shown that cGMP has a major effect on the susceptibility of tsetse flies to establishment of midgut trypanosome infections. There are two systems that the cyclic nucleotide could affect: the trypanosome and/or the tsetse fly. It has been suggested that treatment of malpighian tubules in vitro with cGMP modulates expression of anti-microbial peptides in Drosophila [5] and it has been reported that the antimicrobial peptide, attacin, is involved in trypanosome clearance from tsetse [6]. On the trypanosome side, although the genomes of T. brucei [7], Trypanosoma cruzi [8] and Leishmania major [9] did not show the presence of a typical guanylate cyclase, a cGMP dependent enzyme has been found in Leishmania [10] and a protein kinase has been shown to function through cGMP in T. brucei [11]. Previous studies found guanylate cyclase activity in T. cruzi [12] and it was suggested this activity was involved in cellular motility [13]. More recently guanylate cyclase activity has been found in Leishmania donovani [14].
8-Br-cGMP has been shown to induce both RNA and protein changes in cultured procyclic trypanosomes, indicating that cGMP signalling may be important in trypanosome biology [15]. We have shown that cGMP can "rescue" dying trypanosome infections up to four days after trypanosomes have entered the tsetse fly midgut; by contrast glutathione was only able to rescue such infections within two days post-infection [3], suggesting that the majority of trypanosomes normally die within two days of ingestion.
Unlike the continual feeding of glucosamine to infected tsetse (which decreased rates of trypanosome maturation [16]), in the present work continual feeding of 8-Br-cGMP had no effect on maturation rates of infected tsetse.
In conclusion the current work has shown that the guanylyl cyclic nucleotide, cGMP, increases susceptibility of tsetse flies to trypanosomes. Whether or not this effect works through the fly and/or the trypanosome is as yet unclear.