Tissue-specific expression of esterases in Triatoma infestans (Triatominae, Heteroptera)

Tissue-specific expression of esterases in Triatoma infestans (Triatominae, Heteroptera)

Mara Garcia Tavares^1,3, Maria Tercilia Vilela de Azeredo-Oliveira¹ and Carlos Roberto Ceron²

¹ Departamento de Biologia, ² Departamento de Química e Geociências, Instituto de Biociências, Letras e Ciências Exatas de São José do Rio Preto (IBILCE-UNESP ). Rua Cristóvão Colombo, 2265, Jardim Nazareth, 15054-000 São José do Rio Preto, SP, Brasil. Send correspondence to M.T.V.A.O.

³ Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG, Brasil.

ABSTRACT

We examined the esterases present in the hemolymph and Malpighian tubules of "Kissing bug", Triatoma infestans (Triatominae, Heteroptera) by polyacrylamide gel electrophoresis. Six esterase bands were observed and were designated EST 1 to EST 6. EST 1, 4, 5 and 6 were exclusive to hemolymph, whereas EST 2 and 3 were found only in Malpighian tubules. Each tissue had a characteristic esterase pattern, which may be related to its functional role. The four hemolymph esterases hydrolyzed a-naphthyl acetate. One of these enzymes was classified as a carboxylesterase (EST 4), and another was an acetylesterase (EST 6). The other two enzymes (EST 1 and 5) could be either carboxylesterases or serino-proteases with an esterolytic function, as they were selectively inhibited by phenylmethylsulfonyl fluoride (PMSF). Absence of genetic variability could be due to high inbreeding.

INTRODUCTION

Esterases are a group of highly polymorphic and multifunctional hydrolytic enzymes. Four classes of esterases are now recognized: arylesterases, acetylesterases, carboxylesterases and cholinesterases. Each class has been defined by its substrate specificity, sensitivity to different types of inhibitors, and active site of amino acid residues.

In insects, there is evidence that esterases act on the regulation of juvenile hormone (Kort and Granger, 1981), digestive processes (Kapin and Ahmad, 1980; Jones and Brancoft, 1986), reproduction (Richmond et al., 1980; Mane et al., 1983) and insecticide resistance (Devonshire et al., 1986, 1993; Fournier et al., 1993). Tissue-specific differences in esterase patterns have also been considered. However, functional roles of these enzymes have not yet been established.

We investigated the esterases of hemolymph and Malpighian tubules of "Kissing bug", Triatoma infestans (Triatominae, Heteroptera) in order to obtain information on genetic variability, tissue-specific expression patterns, classification and possible physiological roles.

MATERIAL AND METHODS

Triatoma infestans specimens were provided by the Special Health Service Insectary (SESA), Araraquara, State of São Paulo. All the experimental work was done on adult insects which had been previously fed duck blood.

Hemolymph samples were obtained by extracting a small drop from the ventral region of the insect with a thiny pipette. Malpighian tubules were obtained by dissection. These samples were frozen at -20ºC.

Polyacrylamide gel electrophoresis was performed on individual samples as described by Laemmli (1970), but without SDS. Briefly, 10% concentration slab gels were polymerized in 0.37 M Tris-HCl, pH 8.8. For stacking, 3% gels were prepared in 0.12 M Tris-HCl, pH 6.8. The samples were prepared in 0.0625 M Tris-HCl, pH 6.8, containing 10% glycerol and run at 200 V for 3.5 h at room temperature using a Tris-glycine buffer at pH 8.3. After electrophoresis, gels were soaked for 30 min in 0.1 M phosphate buffer, pH 6.2, in order to reduce pH, and then stained for esterases in 50 ml 0.1 M phosphate buffer containing 60 mg fast blue RR Salt, 15 mg b-naphthyl acetate, 30 mg a-naphthyl acetate and 5 ml n-propanol, at room temperature for 1 h. Silver staining for proteins was carried out using the method of Wray et al. (1981). The protein content of the samples was determined by the Folin-Ciocalteau method (Lowry et al., 1951).

For inhibition analysis, gels were pre-soaked in phosphate buffer (0.1 M, pH 6.2) containing inhibitor for 1 h and then stained for esterase activity as described above, with the inhibitor also present. Inhibitors used were eserine sulfate, malathion, perfection, p-chloromercuribenzoate (pCMB), p-hydroxymercuribenzoate (pOHMB) and phenylmethylsulfonyl fluoride (PMSF). Malathion and perfection were dissolved in a small volume of acetone. pCMB and pOHMB were dissolved in 1 ml of 0.1 M NaOH and PMSF in 1 ml methanol, prior to use. Eserine sulfate was added directly to the pre-soaking and staining solution.

RESULTS AND DISCUSSION

Six esterase band patterns from hemolymph and Malpighian tubules of T. infestans were observed and numbered EST 1 to EST 6, from the fastest to the slowest (Figure 1). Each tissue showed a characteristic esterase pattern. Esterases EST 1, 4, 5 and 6 were found exclusively in hemolymph, whereas EST 2 and 3 were found in Malpighian tubules. This distribution may be related to the functional role of these enzymes. Among hemolymph esterase bands, the largest and generally the most stained was EST 4, followed by EST 5. EST 1 and 6 were less stained. It was also noted that all bands present in hemolymph were a-esterases, because they preferentially hydrolized a-naphthyl acetate. No sex-specific differences were found in hemolymph or Malpighian tubules. EST 2 may be considered b-esterase, because it showed a preference for b-naphthyl acetate. This esterase was always detected as double bands and could be subdivided into EST 2A and 2B which occurred near each other in the gel. On the other hand, EST 3 may be considered to be an a-b-esterase.

Figure 1
- Polyacrylamide gel 10% showing the esterase patterns in Malpighian tubules (MT) and hemolymph (HL) of adult Triatoma infestans male (A) and female (B). Protein content of Malpighian tubule samples varied from 70 to 20 mg while protein content of hemolymph samples varied from 130 to 43 mg in both males and females.

Isozyme polymorphism has been used to characterize populations of triatomines and other insects. We found no qualitative variations in the electrophoretic patterns in spite of the great number of individuals analyzed. Frias and Kattan (1989) reported that domiciliary T. infestans showed less enzymatic polymorphism than wild T. spinolai. These data are supported by Lopez and Moreno's (1995) work, who found greater genetic variability in wild habitats compared to domiciliary habitats in the species Rhodnius prolixus and R. pallescens. Lack of genetic variability in our T. infestans collection may be due to a high inbreeding, as these specimens have been maintained in the laboratory in a closed colony for many generations.

Nine protein bands from T. infestans hemolymph were observed with silver staining (Figure 2A and B). Four were identified as esterases, indicating that almost half of the hemolymph proteins have esterase activity.

Figure 2
- Polyacrylamide gel 10% showing patterns of protein plus esterase bands (A) and esterases only (B) of Triatoma infestans hemolymph (arrows in A indicate esterase bands).

Of the esterases detected in T. infestans hemolymph, EST 1, 4 and 5 were completely inhibited by PMSF (Table I). EST 4 was also inhibited by malathion and less so by perfection. None of these enzymes were affected by eserine or sulfhydryl reagents (pCMB and pOHMB). These observations suggest that among the enzymes found in hemolymph, EST 4 may be a carboxylesterase, containing an active serine, whereas EST 1 and 5 could be trypsin-like enzymes displaying some esterolytic activity, since they were selectively inhibited by PMSF. These observations suggest a proteolytic function for the latter enzymes. EST 6 was slightly inhibited by malathion and perfection and showed resistance to eserine sulfate and sulfhydryl reagents. These results, together with the slight inhibition by PMSF, suggest this enzyme to be an acetylesterase.

Among the esterases detected in the Malpighian tubules, EST 2 is possibly a cholinesterase, because it was highly inhibited by eserine sulfate and organophosphate inhibitors. Furthermore, EST 3 may be a carboxylesterase, because it was inhibited by organophosphates and was resistant to eserine sulfate.

Although cholinesterases are enzymes confined largely to the CNS, cholinesterases with noncholinergic functions have already been described in non-neural tissues (Chatonnet and Lockridge, 1989). Such isozymes are termed nonspecific or pseudocholinesterases. EST 2 identified in Malpighian tubules of T. infestans as a cholinesterase presented some common features with EST 9 and 10 of Drosophila melanogaster, which were classified as pseudocholinesterases (Healy et al., 1991). Villar et al. (1980), analyzing embryonic development of T. infestans, also identified some pseudocholinesterases, acetylcholinesterases, carboxylesterases and aryl plus acetylesterases by substrate specificity and paraoxon inhibition. Based on these data, we suggest that EST 2 of T. infestans represents another example of insect nonspecific cholinesterase which has a noncholinergic function in Malpighian tubules.

ACKNOWLEDGMENTS

The authors are indebted to Dr. José M. Soares Barata, Director of Insectary (Araraquara, SP), Epidemiology Department, Public Health Faculty (São Paulo, SP) and to Bento Gregório de Jesus and João Molina Gil, technicians at the Insectary for providing the insects, and to Dr. James Robert Coleman for reading the manuscript. The support of CNPq and CAPES is gratefully acknowledged. Publication supported by FAPESP.

RESUMO

Foram examinadas as esterases presentes na hemolinfa e nos túbulos de Malpighi do "barbeiro" Triatoma infestans (Triatominae, Heteroptera) através de eletroforese em gel de poliacrilamida. No total, foram observadas seis bandas esterásicas denominadas EST 1 a EST 6. As esterases EST 1, 4, 5 e 6 foram exclusivas da hemolinfa, enquanto EST 2 e 3 foram encontradas apenas nos túbulos de Malpighi. Cada tecido apresentou um padrão esterásico característico, o qual pode estar relacionado com o seu papel funcional. As quatro esterases da hemolinfa hidrolizaram o a-naftil acetato. Uma destas enzimas foi classificada como carboxilesterase (EST 4), uma como acetilesterase (EST 6) e as outras duas enzimas podem ser carboxilesterases ou serino-proteases com função esterolítica, uma vez que elas foram inibidas seletivamente pelo PMSF (EST 1 e 5). A ausência de variabilidade genética pode ser devida à alta taxa de endocruzamentos.

(Received August 14, 1997)

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Publication Dates

• Publication in this collection
  01 Mar 1999
• Date of issue
  Dec 1998

History

• Received
  14 Aug 1997