Spiroplasma species in the Costa Rican highlands: implications for biogeography and biodiversity

Abstract

More than 1,000 Spiroplasma isolates have been obtained from horse flies and deer flies (Diptera:Tabanidae) in the United States and Canada. However, the spiroplasma biota of Central America is poorly known. In August of 1995 and 1998, 13 isolates were obtained in 14 attempts from horse flies of a single species, Poeciloderas quadripunctatus, taken in the Costa Rican highlands (1,100–2,000 m). The majority of the “isolates” proved to be mixtures of two or more Spiroplasma species, but after filter cloning, single strains emerged that were designated as representatives of the 13 accessions. Six distinct spiroplasma serogroups were identified from these isolations. Three of the strains are putative new species with no serological relationship to any other Spiroplasma species. A fourth strain is a putative new species that may be distantly related to S. helicoides, a southeastern U.S. species. These four strains are accorded herein status as representatives of new serogroups: strain BARC 4886 (group XXXV); strain BARC 4900 (group XXXVI); strain BARC 4908 (group XXXVII); and GSU5450 (group XXXVIII). A fifth Spiroplasma species was very closely related to S. lineolae, known previously only from the Georgia (U.S.) coast. The sixth was most closely related to subgroup VIII-3, known from Texas and the southeastern U.S. Discovery of six spiroplasma species in only 13 attempted isolations reflects diversity seldom equaled in southeast Georgia, and never elsewhere in the U.S. These results are consistent with a hypothesis that spiroplasma diversity increases from north (Nova Scotia) to south (Georgia and Costa Rica). The discovery of significant affinity between some spiroplasmas of the southeastern U.S. and the Costa Rican highlands was unexpected, but may reflect a climatically complex Pleistocene history.

Appendices

Appendix 1. Techniques for isolation of spiroplasmas, biochemical tests and serology

Techniques for isolations from the insects, biochemical tests, and serological tests were performed as follows.

Spiroplasma strains

The primary isolates were obtained by standard techniques (Markham et al. 1983; Clark et al. 1984; Wedincamp et al. 1996) from the abdominal viscera of their insect hosts. Briefly, each fly was placed in 30 ml of 0.5% NaOCl plus 0.4 ml of Photo-Flo 200^® (Eastman Kodak) for >45 s, rinsed for >45 s, and blotted on an unbleached paper towel, and the last two abdominal segments were cut off. The viscera were removed with fine-pointed forceps, minced in M1D broth (Whitcomb 1983), passed through filters with 450-nm pores, and then incubated at 30°C.

Culture medium and cultivation techniques

Spiroplasma cultures were maintained at ambient temperature in the field and during air transport to the Statesboro, Georgia, laboratory. After a few passages in M1D broth in Statesboro, all isolates except GSU5450 were transported to the Beltsville, Maryland, laboratory, where they were grown in M1D broth at 30°C and serially subcultured. After 6–12 passages to establish satisfactory growth and to resolve possible mixtures, the strains were triply cloned (Whitcomb et al. 1987) in M1D broth. Cells of the strains growing in M1D broth cultures in logarithmic phase were examined at a magnification of 1,250× by dark-field microscopy during early passages and, later, during the cloning process. Replicated lyophils of the strains were then prepared and stored at -20°C. On the basis of cellular morphology and preliminary serological tests, seven of the strains were selected for antiserum production. The cloned strains were grown in 500 ml of M1D medium, pelleted, and then used as immunogens. Antiserum to six of the strains was prepared in the National Institute of Allergy and Infectious Diseases laboratory in Frederick, Maryland. Antiserum directed against the GSU5450 strain was processed by similar immunologic procedures in the Statesboro laboratory.

Biochemical tests

Tests for utilization of glucose (Aluotto et al. 1970) and arginine (Hackett et al. 1996) were performed as previously described.

Tests for serological cross-reactions

Hyperimmune antisera to all established and putative groups, subgroups, and species (Williamson et al. 1998) of Spiroplasma were taken from the reference collections at the Beltsville Agricultural Research Center and the Frederick laboratory. These antisera and spiroplasmas were then employed in spiroplasma deformation tests (Williamson et al. 1978, 1979, 1998; Whitcomb and Hackett 1996) for screening of the isolates.

Appendix 2. Successive serological identification of Apis Group isolates

(i) Four of the strains (BARC 4901, BARC 4902, BARC 4903, and BARC 4905) reacted at moderate to high titers (1:320) with antiserum to group XXVII Spiroplasma lineolae, a species that has been isolated occasionally from Tabanus lineola from coastal Georgia. (ii) Two of the strains (BARC 4900 and BARC 4904) failed to react with any of the typing sera directed against U.S. spiroplasmas. One of these strains (BARC 4900) was selected for antiserum production. (iii) In early screening, five of the strains (BARC 4886, BARC 4906, BARC 4907, BARC 4908, and GSU5450) showed a low-level (1:40 or 1:80) reaction with antiserum directed against S. helicoides. Following the serological tests, all of these strains were triply cloned, and each was used for antiserum production. In the first cycle of antiserum production, strain BARC 4886 was chosen to represent strains BARC 4886, BARC 4906, BARC 4907, and BARC 4908. The newly prepared antisera were then used in reciprocal crosses among the 13 cloned isolates. Each new antiserum had an intermediate to high level of reactivity (320–2,560) against its antigenic homologue. The preliminary groupings proved to be accurate for strains BARC 4899, BARC 4900, and BARC 4903. Strain BARC 4903 had a high level of reciprocal cross-reactivity (640–1,280) with the TALS-2 strain of S. lineolae. Of the five strains that had exhibited low level reactivities with S. helicoides during early passage, only the antigenic homologue reacted against antiserum prepared to strain BARC 4886. Strains BARC 4906, BARC 4907, and BARC 4908 failed to react with any of the four new antisera. These three strains, like BARC 4886, tended to share a low level of cross-reactivity against S. helicoides. However, the apparent cross-reactivity of strain BARC 4886 with S. helicoides, for whatever reason, disappeared after completion of triple cloning and antiserum production. Similarly, despite a very low-level one-way reaction with S. helicoides, strain GSU5450 was not otherwise cross-reactive with any other known spiroplasma species and is also considered to represent a putative new species. After the first round of antiserum production, strains BARC 4906, BARC 4907, and BARC 4908 were found to be non-reactive with any of the four new antisera (or any other spiroplasma antiserum). Thus, after the first cycle of antiserum production, there was no means by which the synonymy of these three strains could be inferred. So it was necessary to use all three of these strains as immunogens in a second round of antiserum production. It was only after these antisera had been prepared and reciprocal tests had been performed that it became evident that strains BARC 4906, BARC 4907, and BARC 4908 represented a single new taxon. The third round of antiserum production was performed in 1999 in the Statesboro laboratory, in response to the appearance of strain GSU5450, which was isolated from P. quadripunctatus in the Costa Rican highlands in the 1998 expedition. This strain proved to represent a putative new species which, like strains BARC 4906, BARC 4907, and BARC 4908, cross-reacted very marginally with antiserum directed against S. helicoides but is unrelated to other spiroplasmas.