Clogmia albipunctata: a hematofagia sob uma perspectiva exaptativa

Abstract

Clogmia albipunctata is an insect belonging to the order Diptera, family Psychodidae, which has a holometabolous life cycle with immature stages living in semi-aquatic environments. Although this species is not hematophagous, previous studies have shown that this insect can mechanically transmit pathogens, which is an important feature considering its high synanthropy. The suborder Nematocera is a paraphyletic group, encompassing insects that share morphological and physiological features, including dipterans with medical and veterinary importance, allowing phylogenetic analysis among them. As hematophagy emerged several times within the Nematocera group, this instigated studies to understand basic features or adaptations to its development. An evolutive adaptation can be analyzed under two perspectives: the first one concerns about one features shaped by natural selection for your current function. The second concerns about one feature that gives adaptive value to one characteristic, which is not necessarily related to the evolutionary pressures which selected this attribute. This second is known by exaptation. Hematophagous insects have a tight gut physiology control through intestinal pH control and gut enzyme production. Some morphological features are extremely important for the feeding of these insects, such as gut shape and the mouthparts. As C. albipunctata belongs to the Psychodidae family (same family as sandflies, vectors of Leishmania species), these insects are excellent models for physiological and morphological studies about adaptations and exaptations to hematophagy. The aim of this work is to study aspects of C. albipunctata intestinal physiology in comparison to previous studies done with other nematoceran Diptera, through physiological and morphological comparisons, showing exaptative features. Midgut morphological analyses between male and female C. albipunctata and L. longipalpis, A. aegypti and Culex quinquefasciatus was performed by dissection and visual comparison under optic microscopy. In order to determine the gut pH of C. albipunctata maintained in different dietary conditions, we used the vital pH indicators dye Bromothymol Blue and Bromocresol Purple. To study trypsin production after protein ingestion, we performed kinetic assays using the BApNA substrate. To study catalase enzymatic activity we performed a kinetic assay using H2O2 as substrate. To study α-glucosidase activity in the male and female midgut of C. albipunctata, L. longipalpis, A. aegypti and C. quinquefasciatus, we used p-nitrophenyl-α-Dglucopyranoside as substrate. To test the capacity of C. albipunctata gut contents to inhibit the human complement, we performed classic pathway inhibitory assays. We selected proteins that are widely related to hematophagy, from which we performed gene orthology analyzes using the C. albipunctata genome . Our results showed that there is no morphological difference between male and female C. albipunctata midguts, despite. the midguts of males and females of some hematophagous species show morphological differences. There is no difference between trypsin activity in midguts of male and female C. albipunctata , the lowest activity found in the midgut of insects that fed on human inactivated serum. The catalase activity found in males of two hematophagous species is similar to the activity found in male and female C. albipunctata. Among all insects analysed, only L. longipalpis showed a difference between male and female α-glycosidasemidgut activity. For all species analyzed, we observed α-glycosidase enzymatic regionalization, with the greatest activity found in the anterior portion of the midgut. In normal conditions, male and female C. albipunctata exhibit acidic intestinal pH (pH = ~ 6), same condition observed for sandflies and other hematophagous insects. When fed with a protein-rich solution, we observed midgut alkalinisation, with more evident alkalinisation for females. Females of C. albipunctata was responsive to hemolymph of engorged female L. longipalpis. The trypsin gene obtained from C. albipunctata whole genome shows homology with the trypsin type 2 from L. longipalpis. The catalase enzyme is present as a single copy gene in the C. albipunctata genome. No genes corresponding to the 4-HPPD enzyme were identified in the C. albipunctata genome. Our results confirm the initial hypothesis that some adaptations related to hematophagy are already present in non-hematophagous organisms, which supports our idea that most adaptations to the hematophagy process are exaptations, which represent evolutionary changes that do not necessarily change the function of a given character. This concept explains, at least in part, the multiple independent events related to hematophagy in “Nematocera” throughout the evolution process.