Anais da Academia Brasileira de Ciências (2005) 77(3): 405-430 (Annals of the Brazilian Academy of Sciences) ISSN 0001-3765 www.scielo.br/aabc

Oogenesis and development in triatomines: a biochemical approach

GEORGIA C. ATELLA1, KATIA C. GONDIM1, EDNILDO A. MACHADO2 MARCELO N. MEDEIROS2, MÁRIO A.C. SILVA-NETO1 and HATISABURO MASUDA1 1Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Bloco H Universidade Federal do Rio de Janeiro, UFRJ, Av. Bauhinia s/n, Cidade Universitária, Ilha do Fundão 21944-590 Rio de Janeiro, RJ, Brasil 2Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Bloco G Universidade Federal do Rio de Janeiro, UFRJ, Av. Bauhinia s/n, Cidade Universitária, Ilha do Fundão 21944-590 Rio de Janeiro, RJ, Brasil

Manuscript received on March 3, 2005; accepted for publication on March 30, 2005; presented by Lucia Mendonça Previato

ABSTRACT In triatomines, as well as in other , accumulation of is a process in which an extra-ovarian tissue, the body, produces yolk that are packed in the egg. The main , synthesized by the fat body, which is accumulated inside the , is . This process is also known as . There are growing evidences in triatomines that besides fat body the also produces yolk proteins. The way these yolk proteins enter the oocyte will be discussed. Yolk is a complex material composed of proteins, , and other minor components which are packed inside the oocyte in an organized manner. Fertilization triggers embryogenesis, a process where an will develop. During embryogenesis the yolk will be used for the construction of a new individual, the first instar nymph. The challenge for the next decade is to understand how and where these egg proteins are used up together with their non-protein components, in pace with the genetic program of the embryo, which enables differentiation (early phase of embryogenesis) and embryo differentiation (late phase) inside the egg. Key words: , vitellogenesis, embryogenesis, triatomines, Rhodnius prolixus.

INTRODUCTION living organisms. In oviparous organisms, including insects, occurs apart from During the evolution of species a very important the maternal body. Therefore, egg survival relies on achievement, by Mother Nature, was the develop- the utilization of previously stored material for em- ment of multicellular organisms starting from a bryo’s growth. This material, the yolk, is composed unique cell, the fertilized oocyte. This strategy was of proteins, lipids, sugars and other minor compo- adopted by most multicellular organisms and among nents, and is stored in a highly organized manner them we can recognize oviparous and non-oviparous inside the egg. Following fertilization the egg will Dedicated to the memory of Prof. Dr. Herman Lent by give rise to an entire new organism. During devel- initiative of Pedro L. Oliveira, Instituto de Bioquímica Médica. opment, yolk will be gradually used in accordance Universidade Federal do Rio de Janeiro (UFRJ), RJ, Brasil. Correspondence to: Hatisaburo Masuda with the needs of specific cells imposed by the ge- E-mail: [email protected] netic program of the embryo.

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The accumulation of yolk, or vitellogenesis, tures associated with selective endocytosis. These is a process in which extra-ovarian tissues produce were the pioneering reports that were followed by an yolk protein precursors such as vitellogenin (VG) enormous amount of other reports that corroborated the principal protein of the oocyte (Postlethwait and the initial investigations, as reviewed by Telfer et Giorgi 1985, Raikhel and Dhadiala 1992). Due to al. (1982), Raikhel and Dhadiala (1992), and Sni- the heterosynthetic nature of vitellogenesis in in- girevskaya and Raikhel (2004). These studies led sects, their are specialized for the specific to major milestones in our understanding of how accumulation of yolk proteins. Therefore, macromolecules are taken up from surrounding ex- oocytes present a whole set of structures designed tracellular fluids into both mammalian and insect to select, internalize and store specific proteins, such cells. as microvilli, coated pits, coated vesicles and yolk The adaptation for blood feeding behavior, granules. In most insects, a single phospholipogly- among arthropods, evolved independently in sev- coprotein, now named Vitellin (VT) to differentiate eral groups so, during the course of evolution, dif- it from VG, is the main component of the and ferent metabolic adaptations were used to obtain, serves both for embryonic and, in some cases, early digest and use this special source of (Lehane larval development (Postlethwait and Giorgi 1985, 1991). The majority of blood-feeding insects, in- Zhu et al. 1986, Oliveira et al. 1989). cluding triatomines, ingest a large amount of blood Triatomines were the first group of insects in a single meal, comprising several times the ani- where the accumulation of an extra-ovarian protein mal’s own weight (Friend et al. 1965). The diges- by vitellogenic oocytes was reported. The paper of tion of blood brings to the insect a special problem Sir V.B. Wigglesworth in 1943 described the pres- related to the release of an enormous amount of free ence in the egg of Rhodnius prolixus of a pig- heme upon degradation of hemoglobin. Heme is a ment that was originated from a hemolymphatic pre- toxic molecule due to its ability to generate reactive cursor. The pigment was thought to be partially oxygen species (Aft and Mueller 1983, Vincent et al. degraded hemoglobin “katahemoglobin”. The pig- 1988, Vincent 1989, Smith 1990). Thus, heme toxi- ment described by Wigglesworth was isolated fifty city must be counteracted with very efficient defense two years later, and characterized as RHBP (Rhod- systems. In R. prolixus, several lines of defense have nius Heme-Binding Protein) in our laboratory (Oli- been characterized such as Rhodnius Heme-Binding veira et al. 1995), a protein that functions as an an- Protein (RHBP) (Dansa-Petretski et al. 1995); uric tioxidant while in the hemolymph (Dansa-Petretski acid (Souza et al. 1997); hemozoin formation in et al. 1995) and which is accumulated inside the the midgut (Oliveira et al. 1999); and antioxidant oocytes (Machado et al. 1998). The remarkable enzymes (Paes and Oliveira 1999, Paes et al. 2001). paper of Wigglesworth was, in fact, the very first re- The digested blood, on the other hand, also port on the accumulation of an extra-ovarian protein contains a large amount of amino acids and lipids by vitellogenic oocytes, opening the trail that led to that can be used to produce molecules such as Vitel- the characterization of VG uptake by Telfer (1954). logenins (VGs) that are secreted to the hemolymph William Telfer (1960) was the first person to asso- to be taken up by the growing oocytes. The ma- ciate endocytosis with yolk deposition in oocytes jor hemolymph lipoprotein of insects is called lipo- of Hyalophora cecropia. He reported a selective phorin, and its most studied role is the inter organ concentration of hemolymph-borne proteins and, in transport of lipids (Chino et al. 1977, 1981), thus 1961, the route of entry in yolk granules. Soon af- lipids can be carried and delivered to the ter, Roth and Porter (1964) and Roth et al. (1976) (Kawooya et al. 1988). Lipophorin is also stored observed, in oocytes of the mosquito Aedes aegypti, inside the oocytes of moths (Telfer et al. 1991). the presence of coated vesicles, the cellular struc- Besides proteins, carbohydrates and lipids, the

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A Ovary BOvariole

C Trophic cord Trophic Cord

VitellariumOocyte Tropharium

Fig. 1 – The ovary of Rhodnius prolixus: The ovary was dissected at day 5 after blood meal and spread onto a glass slide and photographed. The ovary (A) is composed of two hemi-ovaries that are connected by a common . Each hemi-ovary is composed of seven structures named ovariole. From the hemi-ovary depicted at the right side of Fig. 1A one ovariole was removed to be shown in detail (B). A line drawing of the ovariole is shown in (C), in order to represent the internal structures (After Huebner and Anderson 1972). oocytes of triatomines need other components such 40 to 42 eggs in 15 days after a single blood meal, as heme, , and also mechanical protection thus each ovariole is able to produce approximately of the egg and protection against microorganisms, three eggs, since Rhodnius ovary contains 14 ovari- since embryo development occurs outside maternal oles. The way these eggs are produced will be com- body. Once fertilized, the egg is laid and the em- mented here. The purpose of this review is to bring bryo development is initiated. So, components nec- into discussion the oocyte proteins of triatomines, essary for embryo development such as DNA, pro- especially because these insects feed on blood and teins, lipids, carbohydrates and enzymes must all be use them as substrates to build up their eggs. We packed inside the egg. present a brief survey of the main proteins and try to describe their mode of entry, their accumulation THE OVARIES OF TRIATOMINES inside the oocyte and their use by the growing em- A broad spectrum of studies with hemipterans, in- bryo. Among the proteins that will be commented cluding triatomines, has established that they are are VGs, lipophorin, Rhodnius Heme-binding pro- telotrophic (Bonhag 1955, 1958, Davis 1956, Mas- tein (RHBP) and Rhodnius Calcium-binding Protein ner 1968, Brunt 1971, Huebner and Anderson 1972, (RCBP). The involvement of enzymes such as phos- Schreiner 1977, Couchman and King 1979, Bun- phatases, proteases and kinases in the mobilization ing 1981). Telotrophic ovary is a quite complex of yolk, during embryogenesis, will also be com- structure; as shown in Fig. 1A R. prolixus ovary is mented. composed of two hemi-ovaries. Each hemi-ovary is composed of seven structures named ovarioles VITELLINS AND which are depicted in Fig. 1B. Internal structures VT is derived fromVG, which in most insects is syn- of the ovariole are depicted in Fig. 1C. The cell bi- thesized in the fat body (Pan et al. 1969, Engelmann ology and structures of telotrophic ovaries were re- 1979, Harnish et al. 1982) as a single or multiple viewed by Telfer et al. (1982) and Huebner (1984). precursors that are processed before secretion to the A of R. prolixus is able to produce around hemolymph (Raikhel et al. 1990, Tufail et al. 2004).

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Synthesis and accumulation of yolk proteins are un- origin of the blood has strong influence on R. pro- der hormonal control as reviewed by Hagedorn and lixus oogenesis (Lima Gomes et al. 1990, Valle et Kunkel (1979), Davey (1981), Engelmann (1990), al. 1987, 1992). Raikhel and Dhadiala (1992) and Bellés (2004). In In R. prolixus, juvenile acts directly some insects, complementary synthesis of VGs by on the ovary, controlling the enlargement of inter- the ovaries has been described (Postlethwait et al. follicular channels, a condition known as patency, 1980, Bianchi et al. 1985, Rousset et al. 1988, allowing macromolecules from the hemolymph, Melo et al. 2000). VG expression and secretion such as VG, lipophorin, RHBP etc to reach the sur- to the hemolymph by fat body and uptake by the face of the oocyte. This enlargement is thought to be ovary are regulated at the hormonal and sexual lev- the consequence of Na+/K+ ATPase activity present els. Since the first report on R. prolixus, involv- in follicular cells. This effect is antagonized by ing corpus allatum (juvenile hormone) in the regu- antigonadotropins (Pratt and Davey 1972b, Davey lation of vitellogenesis (Wigglesworth 1936), it has 1981). It has been suggested that the function of become apparent that juvenile hormone influences antigonadotropins is to assure the cessation of vitel- vitellogenesis in the majority of insects (Brookes logenesis. 1969, Engelmann 1970). The replacement of juve- Recently, Medeiros et al. (2002, 2004) have nile hormone following the removal of corpora allata provided evidences that eicosanoids control insect restores vitellogenesis. oogenesis, modulating the levels of cAMP on the Among hemipterans, the process of vitelloge- ovarian follicles. In many vertebrate processes, in- nesis is, in fact, controlled by juvenile hormone as cluding endocytosis, eicosanoids modulate the level demonstrated by Coles (1965) in R. prolixus, and by of second messengers (Smith 1989). Eicosanoids Mundall and Engelmann (1977) in Triatoma pro- receptors are coupled to changes in the intracel- tracta. On the other hand, it was also demonstrated lular levels of cAMP (Stanley-Samuelson and in R. prolixus that oogenesis is inhibited by ecdysone Pedibhotla 1996). So, events controlled by cAMP in a dose-dependent fashion (Garcia et al. 1979). and protein , such as receptor re- In Triatoma protracta and R. prolixus, vitellogene- cycling, vesicle fusion and vesicles recycling can sis is stimulated by mating. The effect of mating be coordinated by eicosanoids (Fallon and Schwartz on egg production of R. prolixus is such that dur- 1988, Griffiths and Hollinshead 1990, Colombo et ing the early gonadotrophic cycle the rate of vitel- al. 1994, Fallon et al. 1994, Mellman 1996, Foti et logenesis is the same in both mated and virgin fe- al. 1997). In R. prolixus, lipoxygenase products males, but at some point onward vitellogenesis is down-regulate cAMP production, thus increasing strongly inhibited (Pratt and Davey 1972a). Mating the rate of yolk uptake while cyclooxygenase prod- activates corpus allatum to produce juvenile hor- ucts upregulate cAMP levels inhibiting yolk accu- mone through ventral nervous cord (Mundall and mulation. In Hyalophora cecropia, it was reported Engelmann 1977, Davey 1987). It is generally ac- that cyclic nucleotides (cAMP and cGMP) induce cepted that in R. prolixus oogenesis is controlled by termination of VG uptake (Wang and Telfer 1996). , being released in response to abdominal Among triatomines R. prolixus is the most stud- stretch during blood feeding (Davey 1987). Besides ied insect with respect to VG and VT. Scarce in- that, it was suggested that stretching alone is not formation is available in other triatomines species enough to induce oogenesis, which is also depen- such as Triatoma infestans and Triatoma protracta. dent on the presence of proteins in the diet (Garcia The native molecular mass of VG of Triatoma pro- and Azambuja 1985). Insects fed with protein-free tracta is 437 kDa (Mundall and Engelmann 1977) diet failed to induce oogenesis although abdominal and 220 kDa for VG and VT of Triatoma infestans stretch had occurred. It was also observed that the (Salomón and Stoka 1986). VT and VG of Triatoma

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infestans show partial immunological identity with gut of the embryo. This fact suggests that part of VG and VT of Panstrongylus megistus and R. pro- the VT content is used for embryonic differentiation lixus (Salomón and Stoka 1986). and another part for nymph differentiation inside and In R. prolixus, VT native molecular mass is 430 outside of the egg. Looking more carefully to the and 260 kDa (Chalaye 1979). The native particles characteristics of VT, it was possible to recognize dissociate in four subunits under denaturing con- and separate three different populations of VT in R. ditions. The molecular mass of the subunits are prolixus (Salerno et al. 2002). These populations 180 kDa, 158 kDa, 50 kDa and 46 kDa (Masuda were named VT1, VT2 and VT3 according to the and Oliveira 1985). Similar estimates for VT sub- order of elution in an ion exchange column. One in- units were obtained by Valle et al. (1987). The VG teresting feature about them is that they seem to be of R. prolixus is synthesized in the fat body as a high segregated inside the oocyte (Salerno, unpublished molecular mass precursors of 205 kDa and 190 kDa data). Another interesting feature about them is that (Valleet al. 1993). VG is secreted to the hemolymph the content of phosphate varies. VT1 is the less and it is taken up by the growing oocytes at the phosphorylated population and the first to be eluted ovary by receptor mediated endocytosis (Oliveira et from the column (Salerno et al. 2002). This varia- al. 1986). Inside the eggs VG is referred to as VT tion can eventually be explained by the presence of to differentiate it from VG from the hemolymph. two enzymes present in R. prolixus oocytes, an acid Despite this nomenclature, slight physical-chemical phosphatase (Nussenzveig et al. 1992) and casein modifications occur on VG molecule during its up- kinase II (CK II) (Silva-Neto and Oliveira 1993). take and storage, and scarce information is available Masuda and Oliveira (1985) have shown the pres- for most insect groups. Upon fertilization,VT which ence of phosphate associated with protein moiety of was previously accumulated inside the oocyte dur- VT, specifically at residue. CK II recognizes ing the process of oogenesis is used by the new em- clusters of phosphorylated residues, so the bryo and ends up with the eclosion of the first instar participation of this enzyme in the modification of nymph (Oliveira et al. 1989). Embryonic develop- phosphorylation status of this protein is a good pos- ment occurs initially as an embryonic differentiation sibility. Melo et al. (2000) demonstrated in R. pro- that is followed by nymphal differentiation inside lixus that epithelial cells derived from large follicles the egg. During the initial phase VT is not signifi- are able to synthesize ovarian VG (O-VG). O-Vg cantly utilized. In R. prolixus around day 10, when elutes at the same position of VT1, suggesting that katatrepsis occurs (Huebner 1984), only 15% of the VT1 is of different origin from other populations. total VT are used (Oliveira et al. 1989). From this Salerno (2001) demonstrated that VG, synthesized point onward, VT is rapidly used by the embryo and at the fat body, gives rise to the VT3 population as at day 15 the first instar nymph hatches. Around consequence of its post-endocytic modification. Re- 50% of the total content of VT are still present in cently, we have collected evidences that VT2 pop- the gut of the new insect, which disappears in the ulation is synthesized by trophocytes (Salerno, un- following five days (Oliveira et al. 1989). So, pos- published data). So, it looks like the different pop- sibly this strategy is used by these insects to improve ulations found inside the R. prolixus oocytes are the their chance to survive in nature, since the first instar result of a concerted effort of different cells such nymphs have approximately five days to find a new as fat body cells, follicular cells and trophocytes. host from where they will suck blood for the first The origin of different populations may explain the time in their lives. differential behavior of each population in ion ex- It was very intriguing the fact that only 15% of change column. total VT are used to build up a new embryo (kata- No significant differences were observed in trepsis). The remaining VT is utilized inside the neutral composition among all different popu-

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lations of VT, but some differences in the glycosila- by follicle cells derived from large follicles (Melo tion status of proteins were observed. The presence et al. 2000). of glucose associated with the oligossacharide of VG purified from the hemolymph and also associ- NON-VITELLIN YOLK PROTEINS ated with the oligossacharide of ovarianVG suggests As VTs can account for 60 to 90% of the protein that VGs from fat body and ovary are secreted after content of the mature oocyte, other oocyte proteins their oligossacharide is processed at the Golgi appa- have been taken out of the spotlight, independent of ratus. The decrease in the content of glucose of VT3 their being also abundant, varying from 1-20% of must be attributed to the post-endocytic processing total proteins. Although VTs are known to serve of VG. Post-endocytic processing of VT has already as source of amino acids, lipids, phosphate, and been reported by Giorgi et al. (1993). The lack of carbohydrates for the growing embryo (Yamashita glucose in the oligossacharide associated with VT2 and Indrasith 1988), non-VT proteins carry other can be attributed to its different origin. non-proteic components to be used by the embryo It is intriguing that in some insects VG is syn- (biliverdin, , , calcium, heme and an thesized in the fat body, in others also by extra amount of amino acids, phosphate and lipids) follicle cells and in the case of Rhodnius also by the as recently reviewed by Masuda et al. (2004). tropharium cells characterizing its triple origin. The timing of O-VG synthesis of R. prolixus resembles that of egg-specific protein of Bombyx mori (Irie RHODNIUS HEME-BINDING PROTEIN (RHBP) and Yamashita 1983). This protein is also synthe- It has recently been discovered that the presence of a sized in follicle cells and appears in the late phase of pink pigment in the eggs of R. prolixus described in oocyte maturation. It is tempting to speculate that the work of Sir V.B. Wigglesworth (1943) as being O-Vg (or VT1) of R. prolixus plays a similar role, partially degraded hemoglobin (“katahemoglobin”), as egg specific protein in Bombyx mori. In this in- is in fact a new protein (Oliveira et al. 1995) now sect, egg-specific protein is located at the outer layer known as Rhodnius Heme-Binding Protein (RHBP). of the oocyte and utilized even before VG (Irie and Wigglesworth also presented data suggesting that Yamashita 1980). a similar phenomenon was occurring in the cattle VT1 is localized at the outer layer of oocyte tick Boophilus microplus, where thousands of deep- (Salerno, unpublished data), so this population of brown colored eggs are laid after a blood meal. VT will possibly be the first one to be used by the The hypothesis of Wigglesworth that heme embryo, similar to what occurs with egg-specific from vertebrate was being recycled by the arthro- protein of Bombyx mori. The proportion of VT1 pods was also adopted by O’Hagan (1974), who population from the total is around 15%, approxi- showed that an amount of heme equivalent to 10% mately the amount used by the embryo up to day 10 of that found in the blood ingested by the tick was after oviposition (Oliveira et al. 1989) when yolk is found in the eggs of this arachnid. However, the enclosed by the embryo (Kelly and Huebner 1986). suggestion that the heme from the diet was being re- A very important feature of Rhodnius VTs is used was not demonstrated directly and was openly the fact that they are produced and stored inside the in disagreement with the well-established concept egg in different periods of oocyte maturation. VT2 that all nucleated cells make their own heme (Ponka is probably the first one to be stored inside the egg 1997). This apparent conflict was directly investi- since it is synthesized by the trophocytes (Coelho gated by Braz et al. (1999), showing Boophilus as and Gomes, personal communication), followed by the first multicellular organism to be unable to make the deposition of VT3 from the hemolymph. The its own heme. Therefore a hallmark in the adapta- last VT to be deposited is VT1 which is synthesized tion of ticks to feed on blood during evolution may

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have been the acquisition of mechanisms to absorb rated with heme and cannot bind additional heme. and transport heme through the hemolymph and de- So, it is not possible to attribute any anti-oxidant liver it to cells, especially to the growing oocytes, role to this protein inside the oocyte. The function where a huge amount of heme is accumulated dur- of RHBP in the egg must be of a different nature from ing oogenesis. that in the hemolymph. The presence of RHBP as- We isolated and characterized the pink pigment sociated with heme in the oocyte is responsible for (RHBP) from both hemolymph and oocytes of R. the characteristic pink color of R. prolixus’ oocytes. prolixus, showing that it was in fact a hemeprotein RHBP is accumulated by growing oocytes composed of a single 15-kDa polypeptide chain. through receptor-mediated endocytosis and is di- cDNA of RHBP was cloned and sequenced. The rected to the yolk granules (Machado et al. 1998). full-length RHBP cDNA codes for a pre-protein of Although several organs and tissues are able to in- 128 amino acids and the deduced amino acids se- corporate RHBP, the ovary is by far the most active quence present no significant homology to any site of RHBP accumulation. known proteins (Paiva-Silva et al. 2002). Studies Limiting the availability of heme-RHBP, but of RHBP structure are under progress (Nagem et maintaining normal levels of VG and lipophorin in al. 2001). the hemolymph, the of R. prolixus produce The presence of apoRHBP in the hemolymph only a few but normal eggs. Experimental maneu- functions as an antioxidant defense to protect Rhod- vers to increase the concentration of heme-RHBP nius from the challenge of dealing with the excess in the hemolymph resulted in an increased num- of heme formed upon hemoglobin digestion. RHBP ber of eggs produced in a dose-dependent manner in the hemolymph promptly binds one heme (Machado et al. 1998). This is an indication that per polypeptide chain non-covalently. The bind- a balanced amount of heme-RHBP and other yolk ing of heme by RHBP suppresses the generation proteins such as VT is required to build up the yolk of activated oxygen species, thus protecting the in- granules. sect against oxidative stress (Dansa-Petretski et al. VG and lipophorin do not compete with RHBP 1995). As mentioned above, in spite of the heme during the process of RHBP uptake, suggesting ei- detoxification mechanisms (hemozoin formation), ther the existence of different receptors or different part of the heme, originated from the digestion of binding sites for the same receptor. Recently, the hemoglobin, crosses the gut wall (Dansa-Petretski et presence of a lipophorin receptor has been discov- al. 1995), and binds to RHBP, which led to the idea ered, independent of the Vg receptor in Aedes ae- that heme was being recycled in Rhodnius. The sit- gypti oocyte (Cheon et al. 2001). Independently of uation in Rhodnius is more complex than that of the the mechanism used for their internalizations, it is tick, because the hemipteran has a fully active heme clear that a cross information is required in order to pathway (Braz et al. 1999). Hence, obtain a balanced amount of RHBP and VG inside possibly Rhodnius synthesize its own heme but also the mature egg. When RHBP is limiting, the strategy recycles part of the heme from the digestion of host used by the female is to reduce the number of viable hemoglobin. eggs. All attempts to induce R. prolixus females to An important function of RHBP in the hemo- produce eggs without heme-RHBP, using diets free lymph is to transport heme to the heart where it is de- of heme, failed (Machado, unpublished data). graded to biliverdin (Paiva-Silva, unpublished data). The expression of RHBP is induced by a blood This “unloading” of RHBP in the heart is probably meal (Paiva-Silva et al. 2002) and is temporally cor- important to help the insect to maintain a safe level related with the appearance in the hemolymph of of apo-RHBP in the hemolymph. other yolk proteins such as VG (Oliveira et al. 1986) RHBP isolated from the oocyte is fully satu- and lipophorin (Gondim et al. 1989), providing the

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balanced need of VG RHBP and lipophorin for egg Hemozoin formation can be promoted by perimi- production. Besides that, RHBP secretion by fat crovillar membranes of the midgut (Oliveira et al. bodies is also dependent on intracellular levels of 2000), but it can also be an autocatalytic process, heme (Paiva-Silva et al. 2002). The amount of heme dependent on the existence of pre-formed hemozoin originated from hemoglobin digestion is not enough (Dorn et al. 1995). The presence of hemozoin in the to guarantee normal oogenesis in vitellogenic Rhod- midgut of the non-fed nymphs will possibly seed the nius females. De novo heme biosynthesis is neces- formation of more hemozoin as the result of blood sary to obtain a normal vitellogenesis. Using suc- digestion that will occur when the insect feeds on cynil acetone, a specific inhibitor of ALA dehy- vertebrate blood for the first time in its life. This is dratase (Lindblad et al. 1977), a substantial decrease also an important reason for the presence of RHBP in in the number of eggs laid is observed (Braz et al. the eggs. Taken together, these observations demon- 2001), which is reversed by feeding the insects with strate that RHBP utilization during embryogenesis porphobilinogen, an intermediate downstream in the can provide heme to de novo synthesis of embryo pathway. hemeproteins. Also that is a source of food for the Very recently, it has been shown that the entry newly hatched nymph and seeds the formation of of RHBP in the oocyte is controlled by eicosanoids hemozoin in the gut of the newly hatched nymph,

(Medeiros et al. 2002, 2004). PGE2 (one of the to prepare the insect to receive its first ingestion products of the cyclooxygenase pathway) decreased of blood in nature. All these events are important RHBP uptake, while indomethacin (a cyclooxyge- enough for the embryo development so that the fe- nase inhibitor) increased uptake in vitro and in vivo. males of R. prolixus do not produce a single egg Upon treatment with indomethacin, two ovarian without RHBP in it. polypeptides (18 and 25 kDa) were dephosphory- lated, suggesting that a protein phosphorylation sig- LIPOPHORIN nal transduction pathway is involved. In fact, there is a growing set of evidences relating endocytosis and During oogenesis in insects, oocytes accumulate a cell signaling pathways (Di Fiore and De Camilli great amount of lipids, which are supplied by lipo- 2001). phorin, a major hemolymphatic lipoprotein (Chino The tight control observed between the uptake et al. 1981, Kawooya and Law 1988, Sun et al. of RHBP and the other yolk proteins such as VT 2000, Ziegler and Ibrahim 2001). Lipophorin trans- required to build up the yolk granules, can be ex- ports different classes of lipids, such as diacylglyc- plained in part by the following observations: a clear erol, phospholipids, , hydrocarbons, free decline in RHBP content is observed during embryo- fatty acids, between insect various tissues, accord- genesis without reduction in heme content (Braz et ing to physiological demand (Soulages and Wells al. 2002), thus suggesting that heme from RHBP is 1994, Blacklock and Ryan 1994, Ryan and Van der recycled. So, a minimum amount of heme, inside Horst 2000). Hematophagous insects, such as the the egg, must be provided in order to obtain a normal triatomines R. prolixus and Panstrongylus megis- development. tus, ingest large amounts of blood at each meal and, In several arthropods including Rhodnius,a like it occurs in other insects, digestion causes the large fraction of the yolk remains unused in the crops release of free fatty acids in midgut lumen which, of first instar nymphs (Oliveira et al. 1989). In the after absorption, are used in the midgut epithe- first five days following hatching, Rhodnius nymphs lium for the synthesis of other lipids, as phospho- completely degrade the remaining yolk proteins and lipids, cholesteryl esthers, tri- and diacylglycerol a dark material can be observed in the lumen of (Tsuchida and Wells 1988, Turunen and Crailsheim midgut of these non-fed insects, possibly hemozoin. 1996, Canavoso et al. 2004, Grillo and Gondim,

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unpublished data). Lipids are then transferred to plied to the ovaries by circulating LDLp (low den- circulating lipophorin that transports them to the or- sity lipophorin), that transfers lipids to the oocytes gans where they are stored and/or utilized (Atella et without protein incorporation (Kawooya and Law al. 1995, Coelho et al. 1997, Canavoso et al. 2004). 1988). In another lepidopteran, Hyalophora ce- In the adult females of R. prolixus, during the cropia, lipophorin is also accumulated by the ovaries days following a blood meal, lipophorin transports (Telfer et al. 1991), and in the mosquito Aedes ae- phospholipids (Gondim et al. 1992) to the ovaries, gypti this lipoprotein can be found in oocytes (Sun and the rate of incorporation of these lipids varies et al. 2000). according to oocyte developmental stage (Gondim So, lipophorin may have its apoproteins incor- et al. 1989), in the same way that it was observed porated or not, depending on insect species and also for the rate of VT uptake (Oliveira et al. 1986). on the organ under analysis, and when lipophorin Considering the fact that during oogenesis a huge is not accumulated by the cells to which it deliv- amount of membranes must be synthesized to cope ers lipids, the particle can be reloaded, acting as a with oocyte growth and accumulation of yolk gran- reusable lipid shuttle (Downer and Chino 1985, Van ules (Telfer et al. 1982), phospholipids incorporated Heusden et al. 1987, Van Antwerpen et al. 1988, by vitellogenic oocytes are possibly used to build up Kawooya and Law 1988). This is the case of the these membranes. In fact, when the fate of radioac- developing ovaries of R. prolixus, where lipophorin tive phospholipids was followed after their transfer apoproteins are not accumulated by the oocytes from lipophorin to oocytes, a great part of radioac- (Gondim et al. 1989, Machado et al. 1996), and tivity was found associated with membranes of yolk it was demonstrated that it can be loaded with lipids bodies (Gondim 1992). at the fat body and the midgut (Atella et al. 1992, Chino et al. (1977) addressed the question of 1995). The midgut shows a greater capacity to trans- lipid accumulation in the ovaries of the silkmoth fer lipids to lipophorin soon after feeding, while the Philosamia cynthia, and concluded that there was fat body shows an increased rate of lipid transfer more lipid in the oocytes than it was expected due later on, when midgut is almost empty (Coelho et to the present amounts of lipophorin and VT. It was al. 1997). proposed, then, that lipophorin delivered lipids, but Although lipophorin is not incorporated by the was not incorporated by the ovaries. developing oocytes of R. prolixus, for delivering In R. prolixus, oocytes store triacylglycerol lipids it interacts with specific binding sites at cell (Santos and Gondim, unpublished data), possibly surface. Receptors for this lipoprotein were studied in lipid droplets, that will be used during embryo- in some insects, as the flight muscles and fat body genesis, as discussed below. So, in triatomines, of Locusta migratoria (Hayakawa 1987, Dantuma lipophorin probably delivers not only phospholipids, et al. 1996), the fat body and midgut of Manduca but also diacylglycerols to the growing oocytes, as it sexta (Tsuchida and Wells 1990, Gondim and Wells does in Manduca sexta, where the participation of a 2000) and the ovaries of Aedes aegypti (Cheon et membrane-associated lipoprotein lipase may be in- al. 2001). In R. prolixus, lipophorin binding to spe- volved (Van Antwerpen et al. 1998). In this moth, cific sites at membranes from fat body and midgut where lipophorin of two density classes (Beenakkers was characterized (Pontes et al. 2002, Grillo et al. et al. 1988) can be found in adult hemolymph, HDLp 2003), and a receptor from oocyte membrane is un- (high density lipophorin) is taken up, associated der investigation. lipids are removed and the lipoprotein is converted Later on, during egg development, imported to a very-high density lipophorin (VHDLp) inside lipids will propel embryo formation. The role the oocytes, and stored there (Kawooya et al. 1988). played by these macromolecules should include fu- However, in this same insect, most lipids are sup- eling of metabolic process, shaping and plasticity

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of cell boundaries, as well as intracellular signaling. Kinsella and Smyth (1966) showed that the Despite the presumed role of lipid and lipid-derived amount of mono- and diacylglycerols remained con- molecules on cell development, the number of stud- stant throughout egg development. However, by ies addressing such matter is scarce. Most of the the time of nymphal emergence the amount of such available studies have focused on non-mammalian lipids increased sharply as a consequence of tria- vertebrates’ models such as , , alli- cylglycerol hydrolysis. Total extractable lipid de- gators and turtles (Rowe et al. 1995, Speake et al. creased from 39.5 to 23.2% of the dry weight of the 1998, Speake and Thompson 1999, 2000). As re- oothecae. Triacylglycerol fraction solely decreased gards insects, most studies have focused on the role by 75%. In the blood sucking-bug R. prolixus a played by lipophorin in lipid storage. Once the num- similar profile was found by our group. Triacyl- ber of studies where triatomine species were em- glycerol content decreased to half the value found ployed as models is still scarce we provide here a in oocytes by the end of egg development period, al- brief account of data including a discussion of sim- though triacylglycerol percentage was kept constant ilar aspects found in other insect orders. during embryogenesis (Santos and Gondim, unpub- The first study providing an analysis on lipid lished data). Free fatty acid (FFA) metabolism was dynamics during insect development was conducted also followed during the same period by Kinsella by Kinsella (1966). In this study a qualitative and (1967). Data indicated a rather different profile with quantitative picture of sterol and sterol esters dy- a surge on FFA content from 0.2 mg% of dry matter namics during Periplaneta americana egg develop- to 1.4% in the last day of egg development. Such ment is depicted. Total sterol content of develop- result is a clear indication of triacylglycerol hydrol- ing is kept constant whereas the propor- ysis mediated by lipase activity particularly before tion of esterifed sterol increased. By the time of nymph hatching. Therefore, measurements of FFA nymphal eclosion the total content of esterified sterol levels are a direct measure of respiratory quotient increased from 20 to 42%. Also, fatty acid compo- once such molecules are catabolized to provide res- sition of the sterol esters is quite altered. Sterol es- piratory energy for both invertebrate and vertebrate ters isolated in early embryogenesis are appreciably tissue (Kinsella 1967). more saturated due to an increase on the content of Dynamics of larval to pupa transition was fol- palmitic (16) and stearic (18) acids. Sterol esters iso- lowed by Pant et al. (1973). Total lipids and neu- lated late on embryogenesis are more unsaturated, tral lipids increased through larval development and due to an increase on the content of oleic (18:1) and attained the maximum concentration at fifth instar (18:2). The significance of such shift on stage. After pupation there was a sudden decrease fatty acid saturation is unclear and is probably linked in pupal fat body lipids, which suggests their uti- to their metabolic function during embryogenesis. A lization on metabolism. FFA and phospholipids are similar shift on the composition of fatty acids asso- also accumulated during the larval development in ciated with neutral lipids was verified by Lipsitz et the insect body. Once after ecdysis a decrease on al. (1969) in the cricket Acheta domesticus. Dur- either free fatty acid or phospholipids is noticed in ing larval development the content of pupal fat body. on monoacylglycerols changed from 34.5% of to- The above mentioned studies employed sepa- tal sterified fatty acids at day 12 to 10.9 at day 30. ration and quantification techniques to follow lipid Such data demonstrate that the profile fatty acid ster- dynamics during insect development. Such strategy ification is probably following punctual adjustments implies in the homogenization of the whole tissue during either embryo/larval development. The role and therefore assumptions could not be made regard- played by such adjustments, as well as the mecha- ing the intracellular localization of such molecules. nism underlying such changes, are still unknown. In order to fulfill this gap, Atella and Shahabuddin

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(2002) employed fluorescently labeled lipids to in- all the metabolic demands of individual cells. Thus vestigate maternal lipid uptake and distribution in calcium is an essential component of the egg, since mosquitoes. Such strategy demonstrated for the first it will be required during development of a new em- time that maternal fatty acid and phospholipids seg- bryo. These observations led to the conclusion that regate differently in tissues of newly hatched mos- this ion must be packed inside the egg during the quito larvae. Inside the adult ovary lipids were found process of oogenesis, in order to allow a normal de- to be stored in yolk granules of developing oocytes velopment of the insect. and distributed evenly. In neonate larvae, however, maternal fatty acids are stored along the side of the Calmodulin (CAM) body. They are located inside structures that resem- One possible way to pack calcium ion inside ble the larval fat body, especially at the base of the the oocyte is through the calcium-binding protein, body hair and in the thorax, where the muscles are lo- calmodulin. Calmodulin (CaM) is a ubiquitous, cated. Such distribution implies a role in energy for acidic, calcium-binding protein of 20 kDa with four- the food intake after . Most maternal phospho- high affinity Ca2+-binding sites present in eukary- lipids are concentrated in the motile intestinal gastric otic cells. When intracellular calcium concentration cecae and are probably released into the gut lumen to rises above 1µM, the binding of calcium drives a act as food emulsifiers during larval feeding. Such conformational change in calmodulin. The calcium- distribution was found in Aedes aegypti and also in bound state of calmodulin associates with a variety Anopheles gambiae suggesting that it may be a con- of proteins and modulates their activities which ex- sequence of a general segregation pathway common plain why it is involved in numerous cellular reg- to several mosquito species. Now, we are testing the ulatory processes. In the cockroach Blatella ger- lipid segregation in R. prolixus embryogenesis. As manica, a large amount of CaM is stored in growing preliminary data show that maternal derived lipids oocytes, where it comprises 0.9% of the total solu- remained in the newborn nymphs, then it is likely bilized protein (Zhang and Kunkel 1992). Isolation that a similar mechanism is taking place in R. pro- of B. germanica CaM followed by detailed molecu- lixus (Fig. 2) (Silva-Neto, Masuda and Atella, un- lar characterization demonstrated close similarity to published data). Fatty acid sterification shift and vertebrate CaMs (Zhang and Kunkel 1992). Small the differential partitioning of lipid classes during structural differences distinguish it from all other egg development described above imply that dur- CaMs. The demonstration of CaM synthe- ing insect egg development a unique and complex sis by vitellogenic follicles, and its absence in the cell biology process is taking place. The extent and hemolymph of the female, together with the demon- uniqueness of such process should receive the at- stration of its synthesis suggest that most of the tention of investigators working in the area in the CaM incorporated by the egg is of ovarian origin. It future. was demonstrated in Oncopeltus fasciatus that a gap junctionally transmitted epithelial cell signal was necessary for yolk uptake (Anderson and Woodruff CALCIUM-BINDING PROTEINS 2001). Similar signal is also necessary in insects Calcium ion is involved in a variety of functions in representing other orders suggesting that possibly living organisms as allosteric activator of some en- this phenomenon was conserved during evolution zymes, modulator of cytoskeletal rearrangements, (Wakmonski and Woodruff 2002). The epithelial muscle contraction, hormone action, membrane fu- cell signal, transmitted through gap junction stimu- sion, transmembrane passage and also as a second lating the incorporation of yolk, is calmodulin messenger. So, the metabolism of calcium must be (Brooks and Woodruff 2004). CaM possibly plays a precisely regulated inside the cells in order to attend dual function: during oogenesis and embryogenesis.

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A B C

Fig. 2 – Maternal lipid localization in R. prolixus neonate nymphs. Purified lipophorin was double labeled with Texas Red PE and ◦ Bodipy FA injected into vitellogenic females; typically four days after injection, eggs were laid. They were kept at 28 C until hatching. Nymphs were immediately collected and kept on ice. Fluorescence associated with newborn nymph was analyzed by fluorescent microscopy. (A) Phase-contrast image. (B) Localization of Bodipy FA in newborn kissing-bug nymph. (C) Localization of Texas Red PE in newborn kissing-bug nymph.

CaM stored in the egg is present in the cytoplasmic tein was isolated from the hemolymph and oocytes compartment surrounding yolk granules and is virtu- of insects fed with blood containing 32Pi, and it was ally absent from yolk granules (Zhang and Kunkel injected into vitellogenic females. Only the ovary 1994). The storage of a huge amount of CaM in was able to incorporate the protein. Both radioac- developing eggs has in turn led to different specula- tive phosphoproteins were detected inside the oocyte tions regarding its role in the programmed degrada- 16 hours following injection. The phosphoprotein 45 tion of VT during embryogenesis. VT is able to bind binds 50 mol of Ca/mol of protein with a K0.5 of CaM and after degradation of VT, the CaM-binding 10-3M. Based on these characteristics, it was named capacity is retained by the 53-kDa fragment gener- Rhodnius calcium-binding phosphoprotein (RCBP). ated by limited proteolysis. Besides that, the amount RCBP resembles phosvitin, one of the most phos- of CaM in developing eggs dramatically decreases phorylated proteins found in nature, isolated from after the beginning of limited proteolysis of the yolk. non-mammalian vertebrates. While phosvitin is a Therefore, CaM can be involved with limited pro- product of the post-endocytic ofVG, RCBP teolysis of VT. The stoichiometry between VT and is not, because it is already present as such in the CaM is (1:1), which reinforces this possibility. hemolymph. As phosvitins (28-35 kDa) are associ- ated with yolk granules (Byrne et al. 1989) it is also Rhodnius Calcium-Binding Protein (RCBP) possible that RCBP is involved with yolk granule In addition to CaM, another calcium-associated pro- formation. tein yolk was isolated and characterized from oocyte The exact function of RCBP in the egg is not and hemolymph from R. prolixus. This protein ca- known, but preliminary data suggest that RCBP is pable of binding calcium and storing it inside the used by developing embryos during the initial 10 oocyte (Silva-Neto et al. 1996) is a phosphoprotein. days of post-oviposition (Silva-Neto, unpublished It exhibits an apparent molecular mass of 18 kDa data) during which katatrepsis is completed and the on gel filtration, but it migrates as an 8 kDa band yolk is enclosed by the embryo (Kelly and Huebner on Tris- SDS-polyacrylamide gels because 1986). The role of RCBP is still not known, and of its high degree of phosphorylation. 24% of the requires further investigation. total number of residues are serine, being phospho- serine the sole phosphorylated amino acid found. YOLK ASSOCIATED ENZYMES A protein that has a similar amino-terminal and re- Female formation in oviparous organisms sembles the oocyte form of the protein was also occurs synchronously with another biochemical found in the hemolymph. Radioactive phosphopro- process which is yolk deposition. Huge amounts

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of carbohydrates, proteins and lipids are gradually jected to freezing and thawing followed by ultra- deposited into growing oocytes during oogenesis. centrifugation. This strategy led to the obtainment Such process will allow embryo development out- of a pellet which contained almost all enzyme activ- side the maternal body. Yolk platelets are special- ity. It also demonstrated a close association between ized organelles where yolk components are stored. the protease and the membrane of the yolk platelet. Nevertheless the composition of yolk platelets has Further experiments showed that R. prolixus (CD) is been already described in detail for many insects a 45 kDa protein whose activity towards a specific but the mechanisms which underlie the utilization fluorescent peptide designed for aspartyl proteases of yolk components, by developing embryo, remain increases over 15-fold during egg development (Fi- to be understood in detail. A recurrent strategy has alho et al. 2005). been the identification and biochemical analysis of A second enzyme associated to yolk platelets yolk platelet-associated enzymes. Such analysis has was a lysosomal acid phosphatase. Such enzyme paved the way for our current understanding of yolk is able to hydrolyze p-nitrophenyl phosphate with platelet physiology in R. prolixus. a peak of activity around pH 5.0. This reaction is R. prolixus egg development is a fertilization strongly blocked in the presence of sodium fluoride triggered event which occurs continuously over a and tartrate but not by p-chloromercuriobenzoate period of 15 days. The major biochemical event, tak- (Nussenzveig et al. 1992). Later on, it was demon- ing place in yolk granules, is the gradual processing strated that Rhodnius acid phosphatase (AP) activity ofVT. Oliveira et al. (1989) showed a continuous al- is mediated by a single enzyme of 94 kDa (Fialho teration in VT molecule following oviposition. The et al. 2002). CD and AP were therefore physically authors divided this process into two phases: limited associated to yolk platelet and we then hypothesized and extensive proteolysis. The first phase spans for that they should play a role in VT proteolysis during 10 days as embryogenesis proceeds while the main egg development. Such hypothesis was investigated VT bands, detected on native-PAGE gels, progres- as follows. sively migrate further into the gel. The second phase Non-mated females of R. prolixus produce and occurs inside the digestive system following day 10 lay non-fertilized eggs (Davey 1967). Such eggs and VT content declines to half of its initial value at were assayed for AP activity and the enzyme ac- hatching. The remaining material is used up by the tivity obtained was compared with the one from first instar nymph during its initial five days of life fertilized eggs homogenized in different days fol- outside the . In the following paragraphs lowing oviposition (Fialho et al. 2002). AP activ- we describe yolk-associated enzymes studied in our ity is 5-fold increased compared to oocytes in the laboratory, whose role was assigned by examination first days following fertilization. In contrast non- of their biochemical properties during egg develop- fertilized but oviposited eggs showed no activation ment. of this enzyme. This result indicated for the first Nussenzveig et al. (1992) were the first to de- time a close link betweenAP activity and oocyte fer- scribe a set of acid hydrolases associated to yolk tilization. Once VT is a phosphorylated molecule, platelet in R. prolixus. The presence of such en- we followed the fate of metabolic labeled phospho- zymes was first detected incubating purified yolk serines with the aid of radioactive inorganic phos- platelets in different pHs, which induced a differ- phate in both set of eggs. Surprisingly, fertilized ential profile of VT proteolysis. Protease activity eggs showed a decrease of 60% of their phospho- towards VT was maximal at pH values between 3.5 serine content. A similar profile was not observed and 4.5. This reaction was solely blocked by the in non-fertilized eggs where a decrease of only 20% classical cathepsin D (CD) inhibitor, pepstatin A, in was detected. Such results were confirmed by the a dose dependent-manner. Yolk platelets were sub- analysis of phosphoserine content with the aid of

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monoclonal raised against phosphoserine lular compartment, i.e. yolk platelets, we hypoth- (Fialho et al. 2002). We next analyzed the ability esized that these enzymes might have a concerted of phosphorylated molecules to inhibit pNPPase ac- action against VT. Fialho et al. (2005) followed the tivity catalyzed by partially purified AP. VT but not fate of yolk-associated CD activity originally de- isolated phosphoserine, mannose 6-phosphate and scribed in R. prolixus oocytes during egg develop- ATP could block such reaction. This result was a ment. They have shown that CD activity, in total egg clear demonstration that VT is a substrate specifi- homogenates, is blocked by the classical aspartyl cally recognized by eggAP.Therefore, in R. prolixus protease inhibitor, pepstatin A. Surprisingly, AP in- egg fertilization triggers a cascade of signaling event hibitors such as NaF, Na+/K+ tartrate and inorganic which culminates in the dephosphorylation ofVT by phosphate also block VT proteolysis. This block- AP. This finding implied that removal of covalently age is not observed when phosphatase in- bound phosphate groups is a part of a programmed hibitors such as vanadate and phenylarsine oxide or pathway for appropriate embryo utilization of yolk levamisole were used in VT proteolysis assay. NaF reserves. Such biochemical pathway appears to be concentrations able to block isolated AP activity do unique to R. prolixus, once B. germanica developing not affect the activity of partially purified CD. There- eggs, where the same phenomenon was investigated, fore, a specific repressor of VT proteolysis should do not present such alterations (Nordin et al. 1990). be dephosphorylated by AP in vivo. The identity of Efforts from many laboratories working world- this molecule should be addressed further. The surge wide with developing eggs of different arthropods of phosphoproteome techniques will allow a close have provided a huge amount of information about look to the transient nature of phosphate groups at- the physical-chemical properties, amino acid and tached to phosphoproteins during egg development gene sequences of several proteases involved in in the future. Combination of such strategy with VT degradation. Some of the mechanisms, which a complete survey on non-proteic phosphorylated regulate VT proteolysis, have been described as molecules able to be dephosphorylated byAP should well. These mechanisms include for instance the provide some detail on the mechanism of CD regu- expression of genes coding for proteases, inacti- lation in this system. In conclusion, the above data vation of protease activity by inhibitory peptides, demonstrated for the first time that AP, besides its or most commonly, pH-induced activation of self- simple hydrolytic function of low molecular weight processing proteases. Proteolytic systems described substrates, plays a role in the regulation of the initial in developing eggs of arthropods usually rely on the steps of protein digestion inside yolk platelets. acidification of YPs. Such mechanism was demon- Dephosphorylation-coupled digestion of pro- strated in several models such as developing eggs teic substrates is a recurrent theme in cell biology. of Xenopus, and tick and therefore its The presence of covalently bound phosphate on VTs correlation to yolk utilization is clearly established was soon recognized as a remarkable feature of those (Fagotto 1990, 1991, Fagotto and Maxfield 1994). proteins (Allerton and Perlmann 1965). Nowadays, Furthermore, the presence of a unique acidification data concerning the physical-chemical properties, system in R. prolixus composed by an H+-PPase and amino acid sequencing and cDNA sequences are also by an H+-ATPase has been recently demon- available for VTs in different insect groups. Thus strated (Motta et al. 2004). Although these studies it became clear that every VT molecule from any have shed light on a recurrent strategy for the mod- organism contains some degree of covalently bound ulation of protease activity, they do not provide any phosphate in its structure (Byrne et al. 1989, Sap- information about the susceptibility of VT molecule pington and Raikhel 1998). Extensive phosphoryla- to the proteolytic attack. tion is particularly found in non-mammalian verte- Once VT, AP and CD share the same intracel- brate VGs within a domain named phosvitin (Byrne

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et al. 1989). Nearly half of its amino acid com- lation. This would allow the interaction of VT with position is made up of serines and most of them cations at certain regions which might be important are phosphorylated. The presence of such motif on for two processes: first, to keep VT in a paracrys- VTs provides an anionic surface where some non- taline state inside the yolk platelet where its “pack- phosphorylated serines are clustered among phos- ing” degree would require a lower number of wa- phorylated serines. This region is a consensus se- ter molecules; second, to prevent the availability of quence for further phosphorylation by a protein ki- cations such as calcium from being able to trigger nase named casein kinase II (CK II). lipid-peroxidation catalyzed by neighboring heme- CK II is a cyclic nucleotide-independent cal- proteins in the yolk platelet such as RHBP. Such cium/calmodulin-insensitive protein kinase (Silva- hypotheses are currently under investigation in our Neto et al. 2002) strongly inhibited in vitro by hep- laboratory. arin and which is able to utilize either ATP or GTP as phosphate donors in the phosphorylation reac- EICOSANOIDS AS LOCAL REGULATORS tion (Pinna and Meggio 1997). The naturally oc- OF OOGENESIS curring consensus sequences of CK II on VTs make The control of insect oogenesis is associated with these proteins a suitable substrate for the identifica- hormones such as juvenile hormone and ecdysone tion of such enzyme on insect eggs. Silva-Neto and (Engelmann 1979, Kunkel and Nordin 1985). These Oliveira (1993) demonstrated the presence of CK II hormones control the synthesis of yolk protein and on fully grown oocytes of R. prolixus. Such enzyme its receptors (Raikhel and Dhadiala 1992); the pa- is 3-fold activated after oocyte fertilization andVT is tency of follicular cells (Sevala and Davey 1989, its main substrate throughout the whole egg develop- Raikhel and O’Lea 1991); and uptake of yolk pro- ment (Fialho et al. 1999). Limited proteolysis takes teins (Stoffolano et al. 1992). The great complexity place on VT molecule in the third day after ovopo- and variety of cells that compose insect ovaries sug- sition. From this point on CK II activity towards gest the participation of local mediators that also VT gradually disappears. Such result demonstrated control and coordinate oogenesis. However, local a specific structural requirement for the recognition mediators which could help in the control of ooge- of Rhodnius VT by CK II (Fialho et al. 1999). Non- nesis are little understood. fertilized eggs do not display this transient CK II Eicosanoids are local lipid mediators produced activation profile. This result was a strong indica- from the oxygenation of C20 polyunsaturated fatty tion that CK II modulation is also included in the acids, most notably arachidonic acid (C 20:4ω-6). programming of egg development. Isolation of such Once released from membrane phospholipids, ara- enzyme allowed a close look into the dynamic of VT chidonate can be oxygenated by prostaglandin G/H phosphorylation by such enzyme (Silva-Neto et al. synthase (cyclooxygenase) or (5-, 8-, 12-, 15-) lipo- 2002). Incubation of purified VT with purified CK xygenases to form prostaglandins (PGs), leuko- II led to the incorporation of 2 mol of phosphate/mol trienes (LTs) or related hydroxy acids, which can VT. However, the total number of phosphorylation play a role as intra or extracellular signals (Smith sites available can be altered by previous incubation 1989, Stanley 2000, Funk 2001). These compounds of VT with alkaline phosphatase. Under such con- regulate many physiological and physiopathological dition limited dephosphorylation increased the total processes in addition to modulating inflammatory number of phosphorylation sites. However, exten- and immunological responses in (Samuel- sive dephosphorylation led to a dramatic decrease son 1983, O‘Neill and Ford-Hutchinson 1993, Mur- on the total phosphorylation sites. Such results led doch et al. 1993, Funk 2001, Pai et al. 2003), but us to speculate that the presence of CK II in yolk their production is not restricted to these , platelets assures the high degree of VT phosphory- as several invertebrate species have been shown to

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produce PGs and LTs (Meijer et al. 1986, Stanley- sis, as seen in other arthropod models (Spaziani et Samuelson et al. 1991, Petzel et al. 1993, Stanley- al. 1993, 1995, Sagi et al. 1995). Samuelson and Pedibhotla 1996, Stanley 2000, Lipoxygenase products are described in the Reddy et al. 2004). reproductive tract of some invertebrates, e.g. four Eicosanoids have several actions in reproduc- starfish species (Meijer et al. 1986), the sea squirt tive biology of vertebrates (Murdoch et al. 1993, Ciona intestinalis, and the surf clam Spisula so- Priddy and Killick 1993, Funk 2001), which have lidissima (Hada et al. 1997). Pagés et al. (1986) encouraged many research groups to study their rel- demonstrated lipoxygenase activity in Drosophila evance in insect . The first report on melanogaster extracts and also the presence of lipo- the biosynthesis of eicosanoids in the insect repro- xygenase-like immunoreactivity in ejaculatory bulb ductive system was in the house cricket Acheta do- of male flies. The reproductive tract of mated fire- mesticus by Destephano and Brady (1977). They brat Thermobia domestica has lipoxygenase activity showed that male testes and seminal vesicles were (Ragab et al. 1987, 1991), and the egg laying be- able to produce PGs, and a PG synthesizing com- havior is dependent of phospholipase A2 activity, plex was transferred from male to female via sper- which seems to be augmented after mating (Bitsch matophore during mating. That study also showed et al. 1995). In triatomines, Medeiros et al. (2004) that PGs have had a stimulating effect on egg-laying showed, by a pharmacological approach, that the behavior. Since then, the prostaglandin biosynthesis lipoxygenase pathways products act as positive ef- has been recognized in reproductive tracts of sev- fectors of yolk uptake. eral insects as Bombyx mori (Yamaja-Setty and Ra- The eicosanoids signaling in vertebrate in- maiah 1979), Teleogryllus commodus (Loher et al. volves cyclic nucleotide modulation. The partici- 1981), Trichoplusia ni (Hagan and Brady 1982), Lo- pation of second messengers involved in eicosanoid custa migratoria (Lange 1984), Musca domestica signaling in insects or even in invertebrates is scarce (Wakayama et al. 1986), Triatoma infestans (Bren- but there are some evidences of modulation by cyclic ner and Bernasconi 1989) and R. prolixus (Medeiros AMP levels in these systems. Meijer et al. (1986) et al. 2002). showed a reduction of cAMP levels in oocytes of However, the model of PG synthesizing com- starfish Methasterias sp. after treatment with 8(R)- plex transfer and egg laying behavior release showed HETE, which induced oocyte maturation. Sagi et to be not conserved, even among the crickets. In al. (1995) demonstrated an increase in cAMP lev- other models such as L. migratoria, Trichoplusi ni, els following treatment of ovarian tissue with PGE2 Musca domestica, PG injections are not able to in- in the prawn Macrobrachium rosembergii. Spaziani duce oviposition (Stanley 2000). It has also been et al. (1995) also showed, in the crayfish Procam- described that there is a transfer of PGs or arachi- barus paeninsulanus, a correlation among ovarian donic acid rather than enzymes in insects such as tissue contraction, PGF2α levels and tissue cAMP

Bombyx mori and Musca domestica during mating levels. The role of PGE2, which was also augmented (Stanley-Samuelson and Pedibhotla 1996, Stanley during oogenesis progression (Spaziani et al. 1993), 2000). The role of PGs in these systems remains remains to be elucidated. Medeiros et al. (2004) to be elucidated, but Loher (1979) showed a net in- showed, in R. prolixus, that cyclooxygenase prod- crease in the egg number in unmated Teleogryllus ucts (inhibitors of yolk uptake) increase the ovar- commodus females after PGE2 injection. Among ian cAMP levels, while the lipoxygenase products triatomines, Medeiros et al. (2002) showed in R. (stimulators of yolk uptake) act in an opposite way prolixus that derivatives of cyclooxygenase pathway decreasing the level of this second messenger (Wang decrease the yolk protein uptake, suggesting that and Telfer 1996). Worth mentioning is that this is these compounds can act as regulators of oogene- the first time that products of both pathways, by co-

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ordinated manner, could be playing roles in an inver- prolixus. Cloning sequencing and expressing recep- tebrate , in an autocrine and/or tors for VT and non-VT yolk proteins will be cru- paracrine way, regulating the rate of yolk deposition. cial to understand the cellular pathway involved with The late 1980s and the 1990s saw many re- the yolk granule assembly, and to give us some clues ports on molecular biology of genes involved with about the role performed by each of the non-VT yolk eicosanoid signaling in mammals, but this is still to proteins during embryogenesis of triatomines. be achieved in invertebrates (Stanley 2000). Thus, The localization of these proteins and recep- the insect genomes (Heckel 2003), trancriptomes tors inside the oocyte but also the characterization of and proteomes of some organs of these animals the organelles to which they are associated must be could bring light to this area, allowing a better un- subject of future research. Although yolk granules derstanding of synthesis, targeting and metabolism seem morphologically very similar, they are proba- of eicosanoids in insect reproductive physiology. bly heterogeneous in composition and play distinct function. Insects initiate their embryonic develop- ment, after fertilization, by nuclei fusion followed CONCLUDING REMARKS by mitotic nuclear division without the correspond- Although VT is the most represented protein in the ing cytoplasmatic division, resulting in a sincitial majority of insect eggs studied, a classical work done blastoderm. Cellularization takes place at the pe- byYamashita and Irie (1980) on B. mori showed that riphery of the egg, and the embryo initiates its de- eggs with noVG in them can produce normal insects. velopment in the cortical area of the egg. Thus, the They demonstrated thatVG orVT is not essential for yolk granules of the cortical area may possibly be egg maturation and embryonic development. Thus, different from those found in the central core. The other proteins can replace the function played by yolk is used gradually, during embryogenesis, sup- VT in normal egg development. In R. prolixus, the plying materials to support metabolic demands of presence of three different populations of VT with growing embryo dictated by the genetic program of different origins, produced at different periods of the insect. As VT can be replaced by other pro- oocyte maturation, suggests that each of them may teins, non-VT proteins seem to be essential for pro- play different roles during embryogenesis. In B. viding specific substrates, such as their associated mori, part of these roles was substituted by different non-protein component such as heme, calcium, iron proteins. Insects adapted to food sources as differ- and lipids for embryo formation. ent as the blood of vertebrates and produce Enzymes of maternal origin (proteases, phos- VGs (lipoglycophosphorylated proteins) which are phatases, kinases and proton-pump enzymes) in- stored inside their eggs. These molecules contain volved with the degradation of yolk can, possibly, be most, but not all, of the basic elements needed to differentially distributed inside the egg. Immunolo- construct an embryo, including amino acids, carbo- calization of enzymes and other non-VT yolk pro- hydrates, and lipids. Non-VT yolk proteins, such teins along with the different populations of VT will as RHBP, RCBP and lipophorin of R. prolixus, play give us a good picture about the organization of the a specialized role. Each insect has its own partic- insect egg and will help us to understand the phys- ular non-VT protein, possibly complementing par- iology of embryo development. The common fact ticular nutritional elements in function of its adap- observed during early embryogenesis, that only part tation to a particular source of food during evolu- of the total content of VT is used along with some tion. Among triatomines information on VT and non-VT proteins, is consistent with the idea of dif- non-VT proteins is very scarce yet and future re- ferent VT populations playing different roles during search must include efforts on the characterization embryogenesis. of these types of molecules in species other than R. As the result of recent studies, it is becom-

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ing clear that yolk granules are not merely digestive mento Científico e Tecnológico (PADCT) and Fun- vesicles but rather sites where a complex metabolism dação Carlos Chagas Filho de Amparo à Pesquisa occurs, finely regulated in relation to both time and do Estado do Rio de Janeiro (FAPERJ). space. The central issue of developmental biol- ogy is the identification of a diversity of cells that RESUMO were once indistinguishable from one another. With Em triatomíneos, assim como em outros insetos, o acú- the progress in the fields of biochemistry, cell biol- mulo de vitelo é um processo no qual um tecido extra- ogy, and molecular biology new technologies be- ovariano, o corpo gorduroso, produz proteínas que são came available to explore these developmen- empacotadas no interior de um ovo. A principal proteína, tal mechanisms. Reliable knowledge of the fate sintetizada pelo corpo gorduroso, que é acumulada no in- map of the embryo and of the cell lineage of sin- terior de um ovócito, é a vitelogenina. Este processo é gle precursor cells has been achieved, in contrast to também conhecido por vitelogênese. Existem crescentes the relative lack of information on several aspects evidências em triatomíneos, que além do corpo gorduroso, of the metabolism of maternally inherited compo- o ovário também produz proteínas de vitelo. A forma nents of insect eggs. Thus, the challenge for the como estas proteínas de vitelo entram nos ovócitos será next decade is to collect enough data to understand aqui comentada. O vitelo é um material complexo com- how and where these egg proteins are used up to- posto por proteínas, lipídeos, carboidratos e outros com- postos minoritários que são empacotados de uma maneira gether with their non-protein components to support organizada no interior dos ovócitos. A fertilização dis- cell differentiation. Besides that, it is also important para a embriogênese, um processo que culmina com o to understand how their degradation is controlled in desenvolvimento do embrião. Durante a embriogênese pace with the genetic program of the embryo, which o vitelo será utilizado para a construção de um novo in- enables cell differentiation (early phase of embryo- divíduo, a ninfa de primeiro estádio. O desafio para a genesis) and embryo differentiation (late phase) in- próxima década é entender onde e como estas proteínas side the egg. de vitelo são utilizadas junto com os seus componentes Finally, it is necessary to recognize that this não protéicos, em compasso com o programa genético do review is a consequence of a lifetime effort by Dr. embrião, que comanda a diferenciação celular (fase inicial Lent who encouraged young Brazilian students and da embriogênese) e diferenciação do embrião (fase final scientists to engage in the study of triatomines. We da embriogênese) no interior do ovo. Palavras-chave: ovogênese, vitelogênese, embriogêne- were all stimulated by reading his papers, books and se, triatomíneos, Rhodnius prolixus. notes, and also by the scientists he trained.

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