Trypsin Genes From Lutzomyia longipalpis 61
Archives of Insect Biochemistry and Physiology October 2007 doi: 10.1002/arch.
stage, expression of Lltryp2 is again reduced to lev-
els somewhat similar to what was detected for em-
bryo (egg) and L1 through L3 (Fig. 5C). Whether
Lltryp2 is regulated by a feeding mechanism or
whether other factors are present in one or more
stages of the sand fly development still needs to
be determined. In addition, we did not detect a
difference in the expression profiles of both tran-
scripts at 72 h PBM when comparing blood meals
with or without L. chagasi. However, as infections
were carried out with the promastigotes stage of
the parasite, it is plausible that different results may
be obtained when infections are performed using
amastigotes forms of L. chagasi.
In the only studies involving New World sand
flies, Mahmood and Borovsky (1992) reported an
increase in the synthesis of trypsin along larval and
pupae development in Lutzomyia anthophora. Ad-
ditionally, in adult females an increase in enzyme
activity, reaching a peak at 72 h PBM was observed
(Mahmood and Borovsky, 1993). These data are
in accordance with our results on the expression
of Lltryp1 and Lltryp2 in L. longipalpis.
Digestive enzymes have been intensively stud-
ied in mosquitoes. In A. aegypti, early and late
trypsins have been characterized (Barillas-Mury and
Wells, 1993; Barillas-Mury et al., 1994; Noriega et
al., 1994, 1996a,b; Noriega and Wells, 1999; Lu et
al., 2006). The early enzyme is expressed in low
amounts immediately following a blood meal,
while the late trypsin is expressed in large amounts
8 to 36 h after blood acquisition (reviewed by
Noriega and Wells, 1999). Previous data (Barillas-
Mury et al., 1995) suggested that the early trypsin
was essential for induction of expression of the late
trypsin gene. Recently, Lu et al. (2006) demon-
strated that despite the differential expression of
both genes in A. aegypti, the early trypsin does not
regulate expression of the late trypsin. In A.
gambiae, seven trypsin genes were identified (Muller
et al., 1995), with most showing constitutive ex-
pression. Additionally, Giannoni et al. (2001) in-
dicated that regulation of trypsin genes in A.
gambiae might be controlled by GATA transcription
factors and regulated by a putative trypsin regula-
tory element.
Our results in L. longipalpis showed the consti-
tutive expression of Lltryp2 and the blood-regulated
expression of Lltryp1. Lltryp1 is absent in unfed flies,
and expression begins at 2 h and peaks at 12 h
after the blood meal. A return to undetectable, pre-
blood-meal levels is seen at 72 h PBM when blood
digestion is completed. Lltryp2 expression is de-
tected in unfed flies. However, it appears to de-
crease after a blood meal. This lower level of
expression is maintained until the end of the blood
digestion, and regained the levels of unfed flies af-
ter 168 h. Interestingly, expression of Lltryp2 also
is detected in males, at levels comparable to unfed
females. The data presented here differ from the
results obtained for the Old World sand fly P.
papatasi (Ramalho-Ortigao et al., 2003). In P.
papatasi, for the four trypsin cDNAs characterized,
a basal expression of all genes in unfed females
was detected. For L. longipalpis, no expression in
Lltryp1 prior to blood feeding was detected. In ad-
dition, there appears to be a correlation between
the expression of Lltryp2 (constitutively expressed)
and Lltyrp1 (blood induced). The levels of expres-
sion of Lltryp2 are reduced as the levels of Lltryp1
are increasing, and later return to pre-blood meal
values as Lltryp1 is shut down. No such relation
was seen between the trypsin transcripts character-
ized from P. papatasi. Nevertheless, when the P.
papatasi and L. longipalpis sequences were phyloge-
netically aligned, the constitutively expressed
trypsin cDNAs from the two sand fly genera,
Lltryp2, Pptryp1, and Pptryp2 clustered within a
single group, whereas the regulated cDNAs, Lltryp1
and Pptryp4, clustered into another.
In relation to other insects, our results bear a
certain degree of similarity to the early and late
trypsin gene expression found in A. aegypti (Barillas-
Mury and Wells, 1993; Noriega et al., 1996) and
with the expression profiles found for trypsin in A.
gambiae (Muller et al., 1995). In mosquitoes and in
L. longipalpis, the constitutive (early) genes are ex-
pressed in non-blood-fed insects. However, in mos-
quitoes the expression of such genes is blocked 24
h after a blood meal and is absent also in males. In
L. longipalpis, this “early” trypsin is never completely
shut off in blood-fed females and is present in