- Email: [email protected]
The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
ASD571_proof ■ 1 June 2014 ■ 1/9
Arthropod Structure & Development xxx (2014) 1e9
55 56 57 58 59 60 61 journal homepage: www.elsevier.com/locate/asd 62 63 64 65 66 67 68 69 70 71 a a b Wiviane Alves de Assis , Juliana Malta , Paulo Filemon P. Pimenta , 72 Marcelo Ramalho-Ortiga ~o c, Gustavo Ferreira Martins a, * Jose 73 a 74 ~o em Biologia Celular e Estrutural, Departamento de Biologia Geral, Universidade Federal de Viçosa (DBG/UFV), s-graduaça Programa de Po rio, Viçosa, Minas Gerais CEP 36570-900, Brazil 75 Campus Universita b ~o Oswaldo Cruz (Fiocruz-MG), Avenida Augusto de Lima, 1715, rio de Entomologia M Laborato edica, Instituto de Pesquisas Ren e Rachou-CPqRR, Fundaça 76 Belo Horizonte, Minas Gerais CEP 30190-002, Brazil 77 c Department of Entomology, Kansas State University (KSU), Manhattan, KS 66506, USA 78 79 80 a r t i c l e i n f o a b s t r a c t 81 82 Article history: The fat body (FB) is responsible for the storage and synthesis of the majority of proteins and metabolites 83 Received 17 February 2014 secreted into the hemolymph. Oenocytes are responsible for lipid processing and detoxification. The FB is 84 Received in revised form distributed throughout the insect body cavity and organized as peripheral and perivisceral portions in 7 May 2014 85 the abdomen, with trophocytes and oenocytes attached to the peripheral portion. Here, we investigated Accepted 13 May 2014 86 the morphology and the subcellular changes in the peripheral and perivisceral FBs and in oenocytes of Available online xxx 87 the sand flies Lutzomyia longipalpis and Phlebotomus papatasi after blood feeding. In L. longipalpis two88 sized oenocytes (small and large) were identified, with both cell types displaying well-developed Keywords: reticular system and smooth endoplasmic reticulum, whereas in P. papatasi, only small cells were 89 Fat body observed. Detailed features of FBs of L. longipalpis and P. papatasi are shared either prior to or after blood 90 Trophocytes feeding. The peripheral and perivisceral FBs responded to blood feeding with the development of Oenocytes 91 glycogen zones and rough endoplasmic reticulum. This study provides the first detailed description of Lutzomyia 92 Phlebotomus the FBs and oenocytes in sand flies, contributing significantly towards are better understanding of the 93 Sand flies biology of such important disease vectors. 94 © 2014 Elsevier Ltd. All rights reserved. 95 96 97 98 99 toxic substances (homeostasis), and participation in the immune 1. Introduction 100 response. Lipid storage is critical for the development of insects, 101 ensuring survival during pupation and during periods when food The fat body (FB) of insects is an organ of mesodermic origin 102 sources are unavailable, as well as providing the yolk components that is comprised of grouped cells forming ribbons or lobes that are 103 that guarantee the successful development of the eggs (Arrese and delimited by a basal lamina. Though the FB is distributed 104 Soulages, 2010). In addition, lipid reserves can also be mobilized throughout the insect body cavity, it is most abundant in the 105 from the FB in response to energy demand of other organs, as in the abdomen. In several holometabolous insects, the FB is divided in 106 case of flight muscles and ovaries (Canavoso et al., 2001). peripheral portion, which is located just below the integument, and 107 In Diptera, the FB is formed by trophocytes, which are cells that the perivisceral portion, which is formed by lobes within the he108 typically have a cytoplasm rich in mitochondria and rough endomocoel. The perivisceral part is either attached or dissociated from plasmic reticulum (rr) (Raikhel and Lea, 1983; Martins and Pimenta, Q1 109 visceral organ (Haunerland and Shirk, 1995; Roma et al., 2010). 110 A variety of metabolic functions have been assigned to the FB, 2008; Martins et al., 2011a). Trophocytes store cytoplasmic lipid 111 including the supply and storage of nutrients, the elimination of droplets (LDs) and protein granules and glycogen. In female 112 mosquitoes, trophocytes change their morphology and biosyn113 thesis capacity in accordance to cell energetic demands (Cardoso * Corresponding author. Tel.: þ55 31 3899 3492; fax: þ55 31 3899 2549. 114 et al., 2010; Martins et al., 2011b). In some insect orders, trophE-mail addresses: [email protected] (W. Alves de Assis), jumalta@gmail. 115 ocytes have been shown to be associated with ectodermal cells, com (J. Malta), pimenta@cpqrr.fiocruz.br (P.F.P. Pimenta), [email protected] 116 ~o), [email protected] (G.F. Martins). (J.M. Ramalho-Ortiga 117 118 http://dx.doi.org/10.1016/j.asd.2014.05.002 119 1467-8039/© 2014 Elsevier Ltd. All rights reserved. Contents lists available at ScienceDirect
Arthropod Structure & Development
The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi
Q4
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
ASD571_proof ■ 1 June 2014 ■ 2/9
2
W. Alves de Assis et al. / Arthropod Structure & Development xxx (2014) 1e9
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD571_proof ■ 1 June 2014 ■ 3/9
W. Alves de Assis et al. / Arthropod Structure & Development xxx (2014) 1e9
3
oenocytes; and in adult dipterans these can be found scattered ~o, 2012). among the trophocytes (Martins and Ramalho-Ortiga Phlebotomine sand flies (Diptera: Psychodidae) are important vectors of many pathogens, including parasites of the genus Leishmania, the causative agents of leishmaniasis, considered one of the most important neglected tropical diseases, and present in 98 countries (Alvar et al., 2012). In spite of the comparative wealth of information available on the morphology and biochemistry of the FBs in several Diptera species (Martins and Pimenta, 2008; Cardoso et al., 2010; Martins et al., 2011a, 2011b), little is known on the FB of sand flies. Further, it is widely accepted that understanding of the cell morphology in insects significantly assists in determining FB function. Thus, here, we provide for the first time a general characterization of the FB and associated oenocytes in Lutzomyia longipalpis and Phlebotomus papatasi, representative sand flies of New and Old World species, respectively. The roles of FBs and oenocytes correlated to their morphology in sand flies are discussed in the present work.
2. Materials and methods L. longipalpis (Jacobina strain) and P. papatasi (Israel strain) sand flies were from colonies maintained in the Department of Entomology at Kansas State University. All adult flies were fed on 20% sucrose ad libitum; FBs were dissected from six-day-old females 45 h post blood meal (fed on anesthetized mice four days after emergence) and from female flies fed only with sugar (20% sucrose). Sand flies were euthanized and washed using ordinary detergent dissolved in tap water. Under stereoscope their wings and legs were removed and the thorax was perforated with entomological needle #20 (Minucie Ento Sphinx, Czech Republic) to allow diffusion of the fixative (glutaraldehyde 2.5%, 0.1 M cacodylate buffer pH7.2). Samples were maintained in fixative solution at 4 C for up to two months. At least 10 fixed carcasses of each species were washed twice in 0.1 M phosphate buffer saline (PBS), dehydrated in a graded series of increasing ethanol (70e100%), then embedded in historesin (Historesin, Leica, Heidelberg, Germany), cut into 3-mm sections, stained with hematoxylin-eosin (HE), and mounted in Eukit (Fluka, St. Louis, MO, USA) medium. Microscopic images were obtained using a Zeiss Primo Star light microscope (Zeiss, Oberkochen, Germany) fitted with an AxioCam ERC5s digital camera (Zeiss). LDs were measured individually from the histological sections of both peripheral and perivisceral trophocytes of the FBs of L. longipalpis and P. papatasi females fed on sugar or blood. Ten LDs from each sand fly species were arbitrarily selected and measured. LD areas were determined considering their boundaries, and using the software Image ProPlus™. Mean values determined for LD of the two cells (peripheral and perivisceral trophocytes) analyzed were compared using t test of Student. Results were deemed significant when p < 0.05. For scanning electron microscopy (SEM), after fixation, the abdomen was opened laterally using a micro scissor (Petrovich ~o Paulo, Brazil), and the visceral organs Instrumentos Cirúrgicos, Sa were removed. The specimens (n ¼ 5 for each species) were washed twice in 0.1 M PBS, dehydrated in an ascending ethanol dilution
Fig. 2. SEM pictures of peripheral fat body of L. longipalpis. [A] Internal view of the dorsal abdominal integument (i) with fat body lobes (lb) attached. [B] A detailed view of the FB lobe (inset Fig. in 2A) showing its irregular surface covered by a basal lamina (arrow). ns- nervous system; t- trachea.
series (70e100%), critical-point dried using CO2 and sputter coated with gold for observation under a SEM LEO 1430VP (Zeiss, Camlise da bridge, England) at the Núcleo de Microscopia e Microana Universidade Federal de Viçosa (NMM-UFV). For transmission electron microscopy (TEM), specimens (n ¼ 6 for each species) were washed in the PBS and postfixed in 1% osmium tetroxide for 1 h in the dark. After post-fixation, fragments of the abdomen were pre-infiltrated in a solution of LR White resin (Electron Microscopy Sciences-EMS, Hatfield, PA, USA) and 100% ethanol (2:1) for 1 h. Subsequently, the samples were embedded in pure resin and maintained at room temperature for 16 h, followed by polymerization in gelatin capsules (EMS) at 60 C for 24 h. Ultrathin sections were placed on copper grids and contrasted for 20 min in 1% aqueous uranyl acetate and lead citrate. The samples were observed and photographed using a SEM Zeiss EM 109 at NMM-UFV. 3. Results In both L. longipalpis and P. papatasi adult females, the FBs are similar in terms of location and general organization. Hence, FBs are distributed throughout the body cavity in these two species, with the abdominal portion being the most developed in the two cases
Fig. 1. Histological sections stained with HE depicting the fat body (FB) in sand fly adult females. [A] Longitudinal section of a P. papatasi sand fly 45 h after a blood meal showing the distribution of the FB in the head (h) and thorax (t). [B] Longitudinal section showing the distribution and the general aspect of FB underneath the integument (i) in the abdomen of L. longipalpis fed on sugar. [C] Transversal section of abdomen of L. longipalpis fed on sugar showing the distribution of the FB. The perivisceral (circled in red) and the peripheral (circled in green) fat body lobes are indicated. Trophocytes (T) in the perivisceral fat body appear rich in lipid droplets (LD), which are more developed than the ones present in the peripheral FB. h- hemocoel; H- heart; i- integument; M muscle; mg- midgut; mt- malpighian tubes; ns- nervous system; o- small oenocytes associated to peripheral fat body; Ovovary.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD571_proof ■ 1 June 2014 ■ 4/9
Fig. 3. TEM pictures of fat body trophocytes in sand fly females. [A] Cells of the peripheral fat body from a sugar fed L. longipalpis with few lipid droplets (LD). [B] Cells of the peripheral fat body of a blood fed L. longipalpis depicting the lipid droplets (LD) and multiple glycogen zones (g). [C] Cell of the perivisceral fat body from a sugar fed P. papatasi showing the nucleus (N) with the nucleolus (*) and clumps of condensed chromatin (arrowheads). Multiple glycogen zones (g) and lipid droplets (LD) are also seen. [D] Cell of a blood fed P. papatasi showing very large glycogen zones (g), lipid droplets (LD) and an also large protein granule (p). Arrowheads- clumps of condensed chromatin; *- nucleolus; bbasal lamina; m- mitochondrium; N- nucleus; is- intercellular space.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD571_proof ■ 1 June 2014 ■ 5/9
Fig. 4. TEM pictures of the trophocytes of peripheral fat body of sugar fed L. longipalpis [A and B] and perivisceral fat body of blood fed females of P. papatasi [CeF]. In [A] Cell cytoplasm portion with a small rough endoplasmic reticulum (rr) and in [B] Cell cytoplasm portion with several mitochondria (m) and vesicle-like structures (v). [C] Trophocyte from a blood fed female exhibiting large lipid droplets (LD) and rough endoplasmic reticulum (rr). [D] Detail of cell cytoplasm of a blood fed female depicting a large rough endoplasmic reticulum (rr) and mitochondrion (m). [E and F] Detailed views of the trophocyte cytoplasm. Note rough endoplasmic reticulum (rr), occurring even associated to a lipid droplet (LD) and to the nuclear envelope (ne). In [F] autophagy also seems to be present as vesicle-like structures (v). m- mitochondrion; N- nucleus; ne- nuclear envelope.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
ASD571_proof ■ 1 June 2014 ■ 6/9
6
W. Alves de Assis et al. / Arthropod Structure & Development xxx (2014) 1e9
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD571_proof ■ 1 June 2014 ■ 7/9
W. Alves de Assis et al. / Arthropod Structure & Development xxx (2014) 1e9
(Fig. 1A, B). The abdominal FB in sand flies are divided in peripheral and perivisceral, with the former being physically associated with the epidermis and consisting of small spaced amorphous lobes (Figs. 1C and 2). However, the perivisceral FB is much more developed, consisting of ribbons of cells that physically contact visceral organs (Fig. 1C). Fat body lobes are surrounded by a basal membrane that isolates the cells of the inner lobe from the direct contact with the hemolymph (Figs. 2A, B and 3A). We were unable to detect the perivisceral FB in sand flies under the SEM analysis as perivisceral lobes were lost during the dissection and processing of samples. The peripheral and perivisceral FBs in L. longipalpis and P. papatasi are composed of trophocytes that contain an abundant number of LDs within their cytoplasms (Figs. 1C, 3A, B and 4C). Under TEM, these LDs and glycogen zones are more visible in perivisceral trophocytes of sugar fed and blood fed sand flies in comparison with the peripheral FB (Figs. 3e4C). The nuclei of trophocytes in either sugar or blood fed L. longipalpis and P. papatasi have predominantly decondensed chromatin, occupying the central region of the cell (Figs. 3 and 4C). The LDs within trophocytes are larger in the perivisceral FB of sugar fed females (36.29 ± 4.07 mm2) in comparison to blood fed females (17.21 ± 1.28 mm2) (p < 0.0001). On the other hand, LDs are not different in the peripheral FB of sugar (13.93 ± 2.11 mm2) or blood fed (16.93 ± 1.79 mm2) females of P. papatasi (p ¼ 0.42). For L. longipalpis, the LDs in the perivisceral (26.80 ± 3.87 mm2 for sugar fed females and 21.89 ± 1.95 mm2 for blood fed females; p ¼ 0.26) and the peripheral FBs of L. longipalpis (15.35 ± 1.32 mm2 for sugar and 15.64 ± 1.67 mm2 for blood fed females; p ¼ 0.89) do not differ according diet. Protein granules were visibly smaller in the L. longipalpis trophocytes (not shown) in comparison to those present in P. papatasi trophocytes (Fig. 3D). After blood feeding, the cytoplasm of the trophocytes in the peripheral and the perivisceral FB of sand flies remain enriched in mitochondria and in rr. However, the rr are much more developed in the blood fed females than in the sand flies fed on sugar only. In addition, we observed that LDs were surrounded by some rr (Fig. 4) in sugar-fed and blood-fed L. longipalpis and P. papatasi. Several structures resembling autophagic vacuoles are present in sand fly trophocytes, in both sugar fed and blood fed females (Fig. 4B and F). Oenocytes were also detected in association with the trophocytes in the peripheral FB of L. longipalpis and P. papatasi. These cells have a homogeneously stained cytoplasm (Fig. 5A, B). Additionally, in L. longipalpis, two oenocytes distinguishable by their size are observed (Figs. 1C and 6A, B). The larger oenocytes are observed along the entire length of the abdomen and are in close association with the ovaries (Fig. 6A). Both the small and the large oenocytes in L. longipalpis and P. papatasi have extensive infoldings of the plasma membrane (Fig. 6C). In micrographs of sectioned cytoplasm such infoldings appear as cross-sectioned structures (Figs. 5D and 6C). The cytoplasm in both cells is also rich in mitochondria and smooth endoplasmic reticulum (sr) (Fig. 5CeD and 6C). 4. Discussion In the present work the microanatomy of the FBs of the vector sand flies L. longipalpis and P. papatasi adult females were
7
described. The abdominal FB in sand flies are divided in peripheral and perivisceral, and this structural separation of L. longipalpis and P. papatasi adult females (including the peripheral and perivisceral portions) has been described for other taxonomic groups, in different developmental stages of insects, including adult stages of Coleoptera (DeLoof and Lagasse, 1970) and Orthoptera (Lauverjat, 1977), larvae of Lepidoptera (Haunerland et al., 1986, 1990; Haunerland and Shirk, 1995; Raman, 2013), and larvae and adults of respective dipterans of Chironomous thummi (Schin et al., 1977) and Drosophila melanogaster (Franchini et al., 2012), respectively. Interestingly, the perivisceral FB is not found in adult mosquitoes; and the peripheral FB forms a continuous layer of cells (Martins and Pimenta, 2008; Martins et al., 2011a, 2011b), reflective of its embryonic mesoderm origin. The biological significance for the absence of the perivisceral FB in adult mosquitoes remains unclear. Before and after blood feeding, the cytoplasm of the trophocytes in the peripheral and the perivisceral FBs of sand flies are enriched in mitochondria and in rr, as previously reported in female Aedes aegypti (Martins and Pimenta, 2008) and Culex quinquefasciatus (Cardoso et al., 2010) mosquitoes. As sand flies need energy for reproductive and other metabolic activities, we expected that the organelles involved in such processes remain abundant after blood feeding. Consistent with this, we noticed a more developed perivisceral portion after blood feeding with a corresponding increase in sub-cellular lipids and polysaccharides suggestive of the involvement of these cells in the metabolic processes for energy demand and before blood intake (Martins et al., 2011b). After blood feeding, the changes in the subcellular aspects of perivisceral trophocytes are more noticeable than in peripheral FB in both L. longipalpis and P. papatasi. This phenomenon of differentiation of FBs in distinct portions has also been seen within other insect species (Schin et al., 1977; Franchini et al., 2012). For instance, in larvae and pupae of Heliothis zea (Lepidoptera), whereas the peripheral FB is responsible for storage, the perivisceral FB is responsible for synthesis (Haunerland et al., 1990; Haunerland and Shirk, 1995). Such distinct functions also are accompanied by changes in ultrastructure, biochemistry, and gene expression pattern (reviewed by Dean et al., 1985). Generally, trophocytes in both the peripheral and in the perivisceral FBs of L. longipalpis and P. papatasi are responsible for storage and biosynthetic activities. In sand flies, the perivisceral FB is more developed than the peripheral FB, with LDs being larger in the former. Combined, these two characteristics suggest that the peripheral FB plays a crucial role in lipid storage in these insects. It has been reported that an increase in the size of LDs is the result of intense lipogenesis taking place in the FB of sugar fed A. aegypti females (Ziegler and Ibrahin, 2001). Here, we were also able to observe lipogenesis in P. papatasi females, and such is more intense in the perivisceral FB than the peripheral FB of sugar fed females. Several structures resembling autophagic vacuoles are present in sand fly trophocytes, in both sugar fed and blood fed females. Autophagy was reported in A. aegypti trophocytes after vitellogenesis (Bryant and Raikhel, 2011), and as in the mosquito, autophagy might also be involved with organelle turnover and vitellogenesis termination in sand flies. The turnover of the protein synthesis machinery and remodeling allow the trophocytes to complete a secretory phase and get ready to switch to lipid and glycogen storage, as they prepare for the next gonotrophic cycle (Raikhel and Lea, 1983; Raikhel, 1986).
Fig. 5. Small oenocytes in the peripheral fat bodies of sand fly adult females fed on blood. [A] Oenocytes (B) are visible as isolated or as clustered cells in association with trophocytes (T) in P. papatasi. [B] TEM picture of an oenocyte attached to trophocytes (T) in L. longipalpis. [C] The Golgi complex (Gc) is seen together with the smooth endoplasmic reticulum (SR) in high magnification of an oenocyte in P. papatasi. [D] Detailed view of the oenocyte showed in “B” with several mitochondria (m), lipid droplets (LD) and the reticular system. i- integument; is- intercellular space between two oenocytes; m- muscles.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD571_proof ■ 1 June 2014 ■ 8/9
Fig. 6. e Large oenocytes (B) of L. longipalpis female. Fat body (FB)- and ovary follicles (ov)-associated oenocytes in a sugar fed female. *- nucleolus; i- integument; n- nucleus. [B] General view of a large oenocyte (and attached trophocyte) of a blood fed female (T). [C] Several invaginations (I) and mitochondria (m) are commonly observed in the cell cortex. cm- cell membrane; T- trophocyte.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ASD571_proof ■ 1 June 2014 ■ 9/9
W. Alves de Assis et al. / Arthropod Structure & Development xxx (2014) 1e9
Oenocytes were detected in association with the trophocytes in the peripheral FB of L. longipalpis and P. papatasi, and these cells have a homogeneously stained cytoplasm as reported for other Diptera (Clark and Dahm, 1973; Martins et al., 2011c). The presence of sr in these cells could be associated with the metabolism and secretion of lipids and the detoxification process, functions usually attributed to oenocytes (see in Martins and Ramalho-Ortig~ ao, 2012 and papers there in). Certain elements of the Golgi complex as well as LDs are also present in the cytoplasm, which had also been reported in other studies (Camargo-Mathias and Caetano, 1996; Gutierrez et al., 2007; Roma et al., 2010). The presence of LDs in the cytoplasm of oenocytes of sand flies is another evidence that these cells are able to provide lipids that are essential for egg maturation during vitellogenesis (Thomsen, 1956; Tobe and Davey, 1974). No ultrastructural differences were observed in the two types of oenocytes before or after blood feeding in sand flies. In several holometabolous insects, such as D. melanogaster, two separate larval and adult oenocytes were reported, each of which is distinct with respect to cell size and number (Makki et al., 2014 and papers there in). In D. melanogaster, larval and adult oenocytes are morphologically distinct ectodermal derivatives with separate developmental origins (Bodenstein, 1950). This dimorphism of the oenocytes is also present in L. longipalpis. In spite of their different sizes, large and small oenocytes in L. longipalpis are similar under TEM; however, the physiological role of these large cells in this autogenous blood feeding species has to be investigated. In recent years, there has been growing concerns towards developing alternative ways to control sand flies as conventional control methods such as insecticides, and even biological control, have not been effective (Sharma and Shing, 2008). Here, the FB and associated oenocytes of the major sand fly vectors L. longipalpis and P. papatasi were investigated by focusing on aspects of histology and ultrastructure of this important organ. The FB is a key organ with multiple functions, ranging from nutrient storage to innate immunity and may provide the means necessary the development of control strategies based on the interruption of the gonotrophic cycle of sand flies. Further studies will help towards making such control approaches achievable. Q3
Uncited references Jackson and Locke, 1989 Acknowledgments
Q2
We would like to acknowledge Dr. Isaac Clement for comments lises, of this manuscript and the Núcleo de Microscopia e Microana Universidade Federal de Viçosa, Minas Gerais for technical assistance. Financial support was provided by CAPES and by NIH grant AI074691 to JMR-O. References lez, I.D., Bern, C., Herrero, M., Desjeux, P., Cano, J., Jannin, J., den Boer, M., Alvar, J., Ve Leishmaniasis Control Team, W.H.O., 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7, e35671. Arrese, E.L., Soulages, J.L., 2010. Insect fat body: energy, metabolism, and regulation. Annu Rev. Entomol. 55, 207e225.
9
Bodenstein, D., 1950. The postembryonic development of Drosophila. In: Demerec, M. (Ed.), Biology of Drosophila. Wiley, New York, pp. 275e367. Bryant, B., Raikhel, A.S., 2011. Programmed autophagy in the fat body of Aedes aegypti is required to maintain egg maturation cycles. PloS One 6, e25502. Camargo-Mathias, M.I., Caetano, F.H., 1996. Ultrastructural cytochemistry of oenocytes of Pachycondyla villosa ants (Hymenoptera: Ponerinae). Acta Microsc. 5A, 6000e6003. Cardoso, A.F., Cres, R.L., Moura, A.S., de Almeida, F., Bijovsky, A.T., 2010. Culex quinquefasciatus vitellogenesis: morphological and biochemical aspects. Mem. Inst. Oswaldo Cruz 105, 254e262. Canavoso, L.E., Jouni, Z.E., Karnas, K.J., Pennington, J.E., Wells, M.A., 2001. Fat metabolism in insects. Annu Rev. Nutr. 21, 23e46. Clark, M.K., Dahm, P.A., 1973. Phenobarbital-induced, membrane-like scrolls in the oenocytes of Musca domestica Linnaeus. J. Cell Biol. 56, 870e875. Dean, R.L., Locke, M., Collins, J.V., 1985. Structure of the fat body. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry, and Pharmacology, third ed. Pergamon Press, Oxford, pp. 155e210. DeLoof, A., Lagasse, A., 1970. Juvenile hormone and the ultra structural properties of the fat body of the adult Colorado potato beetle, Leptinotarsa decemlineata Say. Z. Mikrosk. Anat. Forsh. 106, 439e450. Franchini, A., Mandrioli, M., Franceschi, C., Ottaviani, E., 2012. Morpho-functional changes of fat body in bacteria fed Drosophila melanogaster strains. J. Mol. Hist. 43, 243e251. Gutierrez, E., Wiggins, D., Fielding, B., Gould, A.P., 2007. Specialized hepatocyte-like cells regulate Drosophila lipid metabolism. Nature 445, 275e280. Haunerland, N.H., Shirk, P.D., 1995. Regional and functional differentiation in the insect fat body. Annu. Rev. Entomol. 40, 121e145. Haunerland, N.H., Nair, K.K., Bowers, W.S., 1986. A larval specific lipoprotein: purification and characterization of a blue chromoprotein from Heliothis zea. Biochem. Biophys. Res. Commun. 134, 580e586. Haunerland, N.H., Nair, K.K., Bowers, W.S., 1990. Fat body heterogeneity during development of Heliothis zea. Insect Biochem. 20, 829e837. Jackson, A., Locke, M., 1989. The formation of plasma membrane reticular systems in the oenocytes of an insect. Tissue Cell. 21, 463e473. Lauverjat, S., 1977. L'evolution post imaginale due tissue adipeux femelle de Locusta migratoria et son controle endocrine. Gen. Comp. Endocrinol. 33, 13e34. Makki, R., Cinnamon, E., Gould, A.P., 2014. The development and functions of oenocytes. Annu. Rev. Entomol. 9, 405e425. Martins, G.F., Pimenta, P.F.P., 2008. Structural changes in fat body of Aedes aegypti caused by aging and blood feeding. J. Med. Entomol. 45, 1102e1107. Martins, G.F., Serr~ ao, J.E., Ramalho-Ortig~ ao, M., Pimenta, P.F.P., 2011a. A comparative study of fat body morphology in five mosquito species. Mem. Inst. Oswaldo Cruz 106, 742e747. ~o, M., Pimenta, P.F.P., 2011b. Histochemical Martins, G.F., Serr~ ao, J.E., Ramalho-Ortiga and ultrastructural studies of the mosquito Aedes aegypti fat body: effects of aging and diet type. Microsc. Res. Tech. 74, 1032e1039. ~o, J.E., Fortes-Dias, C.L., RamalhoMartins, G.F., Guedes, B.A.M., Silva, L.M., Serra ~o, M., Pimenta, P.F.P., 2011c. Isolation, primary culture and morphological Ortiga characterization of oenocytes from Aedes aegypti pupae. Tissue Cell 43, 83e90. ~o, J.M., 2012. Oenocytes in insects. Invertebr. Surv. J. 9, Martins, G.F., Ramalho-Ortiga 139e152. Raikhel, A.S., Lea, A.O., 1983. Previtellogenic development and synthesis in the fat body of a mosquito: an ultrastructural and imunocytochemical study. Tissue Cell 15, 281e300. Raikhel, A.S., 1986. Lysosomes in the cessation of vitellogenin secretion by the mosquito fat body; selective degradation of Golgi complexes and secretory granules. Tissue Cell 18, 125e142. Raman, C., 2013. Electron microscopy and immunogold labelling analysis of smart ndez-Vilas, A. (Ed.), Current nanoparticles (storage proteins) in insects. In: Me Microscopy Contributions to Advances in Science and Technology. Roma, G.C., Bueno, O.C., Camargo-Mathias, M.I., 2010. Morpho-physiological analysis of the insect fat body: a review. Micron 41, 395e401. Schin, K., Laufer, H., Carr, E., 1977. Cytochemical and electrophoretic studies of haemoglobin synthesis in the fat body of a midge Chironomous thummi. J. Insect Physiol. 23, 1233e1242. Sharma, U., Singh, S., 2008. Insect vectors of Leishmania: distribution, physiology and their control. J. Vector Borne Dis. 45, 255e272. Thomsen, E., 1956. In: Observations on the Oenocytes of the Adult Calliphora erythrocephala. Meig. Bertil Hanstrom. Zool Papers, pp. 298e306. Tobe, S.S., Davey, K.G., 1974. Autoradiographic study of protein synthesis in abdominal tissues of Glossina austeni. Tissue Cell 6, 255e268. Ziegler, R., Ibrahin, M.M., 2001. Formation of lipid reserves in fat body and eggs of the yellow fever mosquito Aedes aegypti. J. Insect Physiol. 47, 623e627.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
ASD571_proof ■ 1 June 2014 ■ 1/9
Arthropod Structure & Development xxx (2014) 1e9
55 56 57 58 59 60 61 journal homepage: www.elsevier.com/locate/asd 62 63 64 65 66 67 68 69 70 71 a a b Wiviane Alves de Assis , Juliana Malta , Paulo Filemon P. Pimenta , 72 Marcelo Ramalho-Ortiga ~o c, Gustavo Ferreira Martins a, * Jose 73 a 74 ~o em Biologia Celular e Estrutural, Departamento de Biologia Geral, Universidade Federal de Viçosa (DBG/UFV), s-graduaça Programa de Po rio, Viçosa, Minas Gerais CEP 36570-900, Brazil 75 Campus Universita b ~o Oswaldo Cruz (Fiocruz-MG), Avenida Augusto de Lima, 1715, rio de Entomologia M Laborato edica, Instituto de Pesquisas Ren e Rachou-CPqRR, Fundaça 76 Belo Horizonte, Minas Gerais CEP 30190-002, Brazil 77 c Department of Entomology, Kansas State University (KSU), Manhattan, KS 66506, USA 78 79 80 a r t i c l e i n f o a b s t r a c t 81 82 Article history: The fat body (FB) is responsible for the storage and synthesis of the majority of proteins and metabolites 83 Received 17 February 2014 secreted into the hemolymph. Oenocytes are responsible for lipid processing and detoxification. The FB is 84 Received in revised form distributed throughout the insect body cavity and organized as peripheral and perivisceral portions in 7 May 2014 85 the abdomen, with trophocytes and oenocytes attached to the peripheral portion. Here, we investigated Accepted 13 May 2014 86 the morphology and the subcellular changes in the peripheral and perivisceral FBs and in oenocytes of Available online xxx 87 the sand flies Lutzomyia longipalpis and Phlebotomus papatasi after blood feeding. In L. longipalpis two88 sized oenocytes (small and large) were identified, with both cell types displaying well-developed Keywords: reticular system and smooth endoplasmic reticulum, whereas in P. papatasi, only small cells were 89 Fat body observed. Detailed features of FBs of L. longipalpis and P. papatasi are shared either prior to or after blood 90 Trophocytes feeding. The peripheral and perivisceral FBs responded to blood feeding with the development of Oenocytes 91 glycogen zones and rough endoplasmic reticulum. This study provides the first detailed description of Lutzomyia 92 Phlebotomus the FBs and oenocytes in sand flies, contributing significantly towards are better understanding of the 93 Sand flies biology of such important disease vectors. 94 © 2014 Elsevier Ltd. All rights reserved. 95 96 97 98 99 toxic substances (homeostasis), and participation in the immune 1. Introduction 100 response. Lipid storage is critical for the development of insects, 101 ensuring survival during pupation and during periods when food The fat body (FB) of insects is an organ of mesodermic origin 102 sources are unavailable, as well as providing the yolk components that is comprised of grouped cells forming ribbons or lobes that are 103 that guarantee the successful development of the eggs (Arrese and delimited by a basal lamina. Though the FB is distributed 104 Soulages, 2010). In addition, lipid reserves can also be mobilized throughout the insect body cavity, it is most abundant in the 105 from the FB in response to energy demand of other organs, as in the abdomen. In several holometabolous insects, the FB is divided in 106 case of flight muscles and ovaries (Canavoso et al., 2001). peripheral portion, which is located just below the integument, and 107 In Diptera, the FB is formed by trophocytes, which are cells that the perivisceral portion, which is formed by lobes within the he108 typically have a cytoplasm rich in mitochondria and rough endomocoel. The perivisceral part is either attached or dissociated from plasmic reticulum (rr) (Raikhel and Lea, 1983; Martins and Pimenta, Q1 109 visceral organ (Haunerland and Shirk, 1995; Roma et al., 2010). 110 A variety of metabolic functions have been assigned to the FB, 2008; Martins et al., 2011a). Trophocytes store cytoplasmic lipid 111 including the supply and storage of nutrients, the elimination of droplets (LDs) and protein granules and glycogen. In female 112 mosquitoes, trophocytes change their morphology and biosyn113 thesis capacity in accordance to cell energetic demands (Cardoso * Corresponding author. Tel.: þ55 31 3899 3492; fax: þ55 31 3899 2549. 114 et al., 2010; Martins et al., 2011b). In some insect orders, trophE-mail addresses: [email protected] (W. Alves de Assis), jumalta@gmail. 115 ocytes have been shown to be associated with ectodermal cells, com (J. Malta), pimenta@cpqrr.fiocruz.br (P.F.P. Pimenta), [email protected] 116 ~o), [email protected] (G.F. Martins). (J.M. Ramalho-Ortiga 117 118 http://dx.doi.org/10.1016/j.asd.2014.05.002 119 1467-8039/© 2014 Elsevier Ltd. All rights reserved. Contents lists available at ScienceDirect
Arthropod Structure & Development
The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi
Q4
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
ASD571_proof ■ 1 June 2014 ■ 2/9
2
W. Alves de Assis et al. / Arthropod Structure & Development xxx (2014) 1e9
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD571_proof ■ 1 June 2014 ■ 3/9
W. Alves de Assis et al. / Arthropod Structure & Development xxx (2014) 1e9
3
oenocytes; and in adult dipterans these can be found scattered ~o, 2012). among the trophocytes (Martins and Ramalho-Ortiga Phlebotomine sand flies (Diptera: Psychodidae) are important vectors of many pathogens, including parasites of the genus Leishmania, the causative agents of leishmaniasis, considered one of the most important neglected tropical diseases, and present in 98 countries (Alvar et al., 2012). In spite of the comparative wealth of information available on the morphology and biochemistry of the FBs in several Diptera species (Martins and Pimenta, 2008; Cardoso et al., 2010; Martins et al., 2011a, 2011b), little is known on the FB of sand flies. Further, it is widely accepted that understanding of the cell morphology in insects significantly assists in determining FB function. Thus, here, we provide for the first time a general characterization of the FB and associated oenocytes in Lutzomyia longipalpis and Phlebotomus papatasi, representative sand flies of New and Old World species, respectively. The roles of FBs and oenocytes correlated to their morphology in sand flies are discussed in the present work.
2. Materials and methods L. longipalpis (Jacobina strain) and P. papatasi (Israel strain) sand flies were from colonies maintained in the Department of Entomology at Kansas State University. All adult flies were fed on 20% sucrose ad libitum; FBs were dissected from six-day-old females 45 h post blood meal (fed on anesthetized mice four days after emergence) and from female flies fed only with sugar (20% sucrose). Sand flies were euthanized and washed using ordinary detergent dissolved in tap water. Under stereoscope their wings and legs were removed and the thorax was perforated with entomological needle #20 (Minucie Ento Sphinx, Czech Republic) to allow diffusion of the fixative (glutaraldehyde 2.5%, 0.1 M cacodylate buffer pH7.2). Samples were maintained in fixative solution at 4 C for up to two months. At least 10 fixed carcasses of each species were washed twice in 0.1 M phosphate buffer saline (PBS), dehydrated in a graded series of increasing ethanol (70e100%), then embedded in historesin (Historesin, Leica, Heidelberg, Germany), cut into 3-mm sections, stained with hematoxylin-eosin (HE), and mounted in Eukit (Fluka, St. Louis, MO, USA) medium. Microscopic images were obtained using a Zeiss Primo Star light microscope (Zeiss, Oberkochen, Germany) fitted with an AxioCam ERC5s digital camera (Zeiss). LDs were measured individually from the histological sections of both peripheral and perivisceral trophocytes of the FBs of L. longipalpis and P. papatasi females fed on sugar or blood. Ten LDs from each sand fly species were arbitrarily selected and measured. LD areas were determined considering their boundaries, and using the software Image ProPlus™. Mean values determined for LD of the two cells (peripheral and perivisceral trophocytes) analyzed were compared using t test of Student. Results were deemed significant when p < 0.05. For scanning electron microscopy (SEM), after fixation, the abdomen was opened laterally using a micro scissor (Petrovich ~o Paulo, Brazil), and the visceral organs Instrumentos Cirúrgicos, Sa were removed. The specimens (n ¼ 5 for each species) were washed twice in 0.1 M PBS, dehydrated in an ascending ethanol dilution
Fig. 2. SEM pictures of peripheral fat body of L. longipalpis. [A] Internal view of the dorsal abdominal integument (i) with fat body lobes (lb) attached. [B] A detailed view of the FB lobe (inset Fig. in 2A) showing its irregular surface covered by a basal lamina (arrow). ns- nervous system; t- trachea.
series (70e100%), critical-point dried using CO2 and sputter coated with gold for observation under a SEM LEO 1430VP (Zeiss, Camlise da bridge, England) at the Núcleo de Microscopia e Microana Universidade Federal de Viçosa (NMM-UFV). For transmission electron microscopy (TEM), specimens (n ¼ 6 for each species) were washed in the PBS and postfixed in 1% osmium tetroxide for 1 h in the dark. After post-fixation, fragments of the abdomen were pre-infiltrated in a solution of LR White resin (Electron Microscopy Sciences-EMS, Hatfield, PA, USA) and 100% ethanol (2:1) for 1 h. Subsequently, the samples were embedded in pure resin and maintained at room temperature for 16 h, followed by polymerization in gelatin capsules (EMS) at 60 C for 24 h. Ultrathin sections were placed on copper grids and contrasted for 20 min in 1% aqueous uranyl acetate and lead citrate. The samples were observed and photographed using a SEM Zeiss EM 109 at NMM-UFV. 3. Results In both L. longipalpis and P. papatasi adult females, the FBs are similar in terms of location and general organization. Hence, FBs are distributed throughout the body cavity in these two species, with the abdominal portion being the most developed in the two cases
Fig. 1. Histological sections stained with HE depicting the fat body (FB) in sand fly adult females. [A] Longitudinal section of a P. papatasi sand fly 45 h after a blood meal showing the distribution of the FB in the head (h) and thorax (t). [B] Longitudinal section showing the distribution and the general aspect of FB underneath the integument (i) in the abdomen of L. longipalpis fed on sugar. [C] Transversal section of abdomen of L. longipalpis fed on sugar showing the distribution of the FB. The perivisceral (circled in red) and the peripheral (circled in green) fat body lobes are indicated. Trophocytes (T) in the perivisceral fat body appear rich in lipid droplets (LD), which are more developed than the ones present in the peripheral FB. h- hemocoel; H- heart; i- integument; M muscle; mg- midgut; mt- malpighian tubes; ns- nervous system; o- small oenocytes associated to peripheral fat body; Ovovary.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD571_proof ■ 1 June 2014 ■ 4/9
Fig. 3. TEM pictures of fat body trophocytes in sand fly females. [A] Cells of the peripheral fat body from a sugar fed L. longipalpis with few lipid droplets (LD). [B] Cells of the peripheral fat body of a blood fed L. longipalpis depicting the lipid droplets (LD) and multiple glycogen zones (g). [C] Cell of the perivisceral fat body from a sugar fed P. papatasi showing the nucleus (N) with the nucleolus (*) and clumps of condensed chromatin (arrowheads). Multiple glycogen zones (g) and lipid droplets (LD) are also seen. [D] Cell of a blood fed P. papatasi showing very large glycogen zones (g), lipid droplets (LD) and an also large protein granule (p). Arrowheads- clumps of condensed chromatin; *- nucleolus; bbasal lamina; m- mitochondrium; N- nucleus; is- intercellular space.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD571_proof ■ 1 June 2014 ■ 5/9
Fig. 4. TEM pictures of the trophocytes of peripheral fat body of sugar fed L. longipalpis [A and B] and perivisceral fat body of blood fed females of P. papatasi [CeF]. In [A] Cell cytoplasm portion with a small rough endoplasmic reticulum (rr) and in [B] Cell cytoplasm portion with several mitochondria (m) and vesicle-like structures (v). [C] Trophocyte from a blood fed female exhibiting large lipid droplets (LD) and rough endoplasmic reticulum (rr). [D] Detail of cell cytoplasm of a blood fed female depicting a large rough endoplasmic reticulum (rr) and mitochondrion (m). [E and F] Detailed views of the trophocyte cytoplasm. Note rough endoplasmic reticulum (rr), occurring even associated to a lipid droplet (LD) and to the nuclear envelope (ne). In [F] autophagy also seems to be present as vesicle-like structures (v). m- mitochondrion; N- nucleus; ne- nuclear envelope.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
ASD571_proof ■ 1 June 2014 ■ 6/9
6
W. Alves de Assis et al. / Arthropod Structure & Development xxx (2014) 1e9
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD571_proof ■ 1 June 2014 ■ 7/9
W. Alves de Assis et al. / Arthropod Structure & Development xxx (2014) 1e9
(Fig. 1A, B). The abdominal FB in sand flies are divided in peripheral and perivisceral, with the former being physically associated with the epidermis and consisting of small spaced amorphous lobes (Figs. 1C and 2). However, the perivisceral FB is much more developed, consisting of ribbons of cells that physically contact visceral organs (Fig. 1C). Fat body lobes are surrounded by a basal membrane that isolates the cells of the inner lobe from the direct contact with the hemolymph (Figs. 2A, B and 3A). We were unable to detect the perivisceral FB in sand flies under the SEM analysis as perivisceral lobes were lost during the dissection and processing of samples. The peripheral and perivisceral FBs in L. longipalpis and P. papatasi are composed of trophocytes that contain an abundant number of LDs within their cytoplasms (Figs. 1C, 3A, B and 4C). Under TEM, these LDs and glycogen zones are more visible in perivisceral trophocytes of sugar fed and blood fed sand flies in comparison with the peripheral FB (Figs. 3e4C). The nuclei of trophocytes in either sugar or blood fed L. longipalpis and P. papatasi have predominantly decondensed chromatin, occupying the central region of the cell (Figs. 3 and 4C). The LDs within trophocytes are larger in the perivisceral FB of sugar fed females (36.29 ± 4.07 mm2) in comparison to blood fed females (17.21 ± 1.28 mm2) (p < 0.0001). On the other hand, LDs are not different in the peripheral FB of sugar (13.93 ± 2.11 mm2) or blood fed (16.93 ± 1.79 mm2) females of P. papatasi (p ¼ 0.42). For L. longipalpis, the LDs in the perivisceral (26.80 ± 3.87 mm2 for sugar fed females and 21.89 ± 1.95 mm2 for blood fed females; p ¼ 0.26) and the peripheral FBs of L. longipalpis (15.35 ± 1.32 mm2 for sugar and 15.64 ± 1.67 mm2 for blood fed females; p ¼ 0.89) do not differ according diet. Protein granules were visibly smaller in the L. longipalpis trophocytes (not shown) in comparison to those present in P. papatasi trophocytes (Fig. 3D). After blood feeding, the cytoplasm of the trophocytes in the peripheral and the perivisceral FB of sand flies remain enriched in mitochondria and in rr. However, the rr are much more developed in the blood fed females than in the sand flies fed on sugar only. In addition, we observed that LDs were surrounded by some rr (Fig. 4) in sugar-fed and blood-fed L. longipalpis and P. papatasi. Several structures resembling autophagic vacuoles are present in sand fly trophocytes, in both sugar fed and blood fed females (Fig. 4B and F). Oenocytes were also detected in association with the trophocytes in the peripheral FB of L. longipalpis and P. papatasi. These cells have a homogeneously stained cytoplasm (Fig. 5A, B). Additionally, in L. longipalpis, two oenocytes distinguishable by their size are observed (Figs. 1C and 6A, B). The larger oenocytes are observed along the entire length of the abdomen and are in close association with the ovaries (Fig. 6A). Both the small and the large oenocytes in L. longipalpis and P. papatasi have extensive infoldings of the plasma membrane (Fig. 6C). In micrographs of sectioned cytoplasm such infoldings appear as cross-sectioned structures (Figs. 5D and 6C). The cytoplasm in both cells is also rich in mitochondria and smooth endoplasmic reticulum (sr) (Fig. 5CeD and 6C). 4. Discussion In the present work the microanatomy of the FBs of the vector sand flies L. longipalpis and P. papatasi adult females were
7
described. The abdominal FB in sand flies are divided in peripheral and perivisceral, and this structural separation of L. longipalpis and P. papatasi adult females (including the peripheral and perivisceral portions) has been described for other taxonomic groups, in different developmental stages of insects, including adult stages of Coleoptera (DeLoof and Lagasse, 1970) and Orthoptera (Lauverjat, 1977), larvae of Lepidoptera (Haunerland et al., 1986, 1990; Haunerland and Shirk, 1995; Raman, 2013), and larvae and adults of respective dipterans of Chironomous thummi (Schin et al., 1977) and Drosophila melanogaster (Franchini et al., 2012), respectively. Interestingly, the perivisceral FB is not found in adult mosquitoes; and the peripheral FB forms a continuous layer of cells (Martins and Pimenta, 2008; Martins et al., 2011a, 2011b), reflective of its embryonic mesoderm origin. The biological significance for the absence of the perivisceral FB in adult mosquitoes remains unclear. Before and after blood feeding, the cytoplasm of the trophocytes in the peripheral and the perivisceral FBs of sand flies are enriched in mitochondria and in rr, as previously reported in female Aedes aegypti (Martins and Pimenta, 2008) and Culex quinquefasciatus (Cardoso et al., 2010) mosquitoes. As sand flies need energy for reproductive and other metabolic activities, we expected that the organelles involved in such processes remain abundant after blood feeding. Consistent with this, we noticed a more developed perivisceral portion after blood feeding with a corresponding increase in sub-cellular lipids and polysaccharides suggestive of the involvement of these cells in the metabolic processes for energy demand and before blood intake (Martins et al., 2011b). After blood feeding, the changes in the subcellular aspects of perivisceral trophocytes are more noticeable than in peripheral FB in both L. longipalpis and P. papatasi. This phenomenon of differentiation of FBs in distinct portions has also been seen within other insect species (Schin et al., 1977; Franchini et al., 2012). For instance, in larvae and pupae of Heliothis zea (Lepidoptera), whereas the peripheral FB is responsible for storage, the perivisceral FB is responsible for synthesis (Haunerland et al., 1990; Haunerland and Shirk, 1995). Such distinct functions also are accompanied by changes in ultrastructure, biochemistry, and gene expression pattern (reviewed by Dean et al., 1985). Generally, trophocytes in both the peripheral and in the perivisceral FBs of L. longipalpis and P. papatasi are responsible for storage and biosynthetic activities. In sand flies, the perivisceral FB is more developed than the peripheral FB, with LDs being larger in the former. Combined, these two characteristics suggest that the peripheral FB plays a crucial role in lipid storage in these insects. It has been reported that an increase in the size of LDs is the result of intense lipogenesis taking place in the FB of sugar fed A. aegypti females (Ziegler and Ibrahin, 2001). Here, we were also able to observe lipogenesis in P. papatasi females, and such is more intense in the perivisceral FB than the peripheral FB of sugar fed females. Several structures resembling autophagic vacuoles are present in sand fly trophocytes, in both sugar fed and blood fed females. Autophagy was reported in A. aegypti trophocytes after vitellogenesis (Bryant and Raikhel, 2011), and as in the mosquito, autophagy might also be involved with organelle turnover and vitellogenesis termination in sand flies. The turnover of the protein synthesis machinery and remodeling allow the trophocytes to complete a secretory phase and get ready to switch to lipid and glycogen storage, as they prepare for the next gonotrophic cycle (Raikhel and Lea, 1983; Raikhel, 1986).
Fig. 5. Small oenocytes in the peripheral fat bodies of sand fly adult females fed on blood. [A] Oenocytes (B) are visible as isolated or as clustered cells in association with trophocytes (T) in P. papatasi. [B] TEM picture of an oenocyte attached to trophocytes (T) in L. longipalpis. [C] The Golgi complex (Gc) is seen together with the smooth endoplasmic reticulum (SR) in high magnification of an oenocyte in P. papatasi. [D] Detailed view of the oenocyte showed in “B” with several mitochondria (m), lipid droplets (LD) and the reticular system. i- integument; is- intercellular space between two oenocytes; m- muscles.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
ASD571_proof ■ 1 June 2014 ■ 8/9
Fig. 6. e Large oenocytes (B) of L. longipalpis female. Fat body (FB)- and ovary follicles (ov)-associated oenocytes in a sugar fed female. *- nucleolus; i- integument; n- nucleus. [B] General view of a large oenocyte (and attached trophocyte) of a blood fed female (T). [C] Several invaginations (I) and mitochondria (m) are commonly observed in the cell cortex. cm- cell membrane; T- trophocyte.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ASD571_proof ■ 1 June 2014 ■ 9/9
W. Alves de Assis et al. / Arthropod Structure & Development xxx (2014) 1e9
Oenocytes were detected in association with the trophocytes in the peripheral FB of L. longipalpis and P. papatasi, and these cells have a homogeneously stained cytoplasm as reported for other Diptera (Clark and Dahm, 1973; Martins et al., 2011c). The presence of sr in these cells could be associated with the metabolism and secretion of lipids and the detoxification process, functions usually attributed to oenocytes (see in Martins and Ramalho-Ortig~ ao, 2012 and papers there in). Certain elements of the Golgi complex as well as LDs are also present in the cytoplasm, which had also been reported in other studies (Camargo-Mathias and Caetano, 1996; Gutierrez et al., 2007; Roma et al., 2010). The presence of LDs in the cytoplasm of oenocytes of sand flies is another evidence that these cells are able to provide lipids that are essential for egg maturation during vitellogenesis (Thomsen, 1956; Tobe and Davey, 1974). No ultrastructural differences were observed in the two types of oenocytes before or after blood feeding in sand flies. In several holometabolous insects, such as D. melanogaster, two separate larval and adult oenocytes were reported, each of which is distinct with respect to cell size and number (Makki et al., 2014 and papers there in). In D. melanogaster, larval and adult oenocytes are morphologically distinct ectodermal derivatives with separate developmental origins (Bodenstein, 1950). This dimorphism of the oenocytes is also present in L. longipalpis. In spite of their different sizes, large and small oenocytes in L. longipalpis are similar under TEM; however, the physiological role of these large cells in this autogenous blood feeding species has to be investigated. In recent years, there has been growing concerns towards developing alternative ways to control sand flies as conventional control methods such as insecticides, and even biological control, have not been effective (Sharma and Shing, 2008). Here, the FB and associated oenocytes of the major sand fly vectors L. longipalpis and P. papatasi were investigated by focusing on aspects of histology and ultrastructure of this important organ. The FB is a key organ with multiple functions, ranging from nutrient storage to innate immunity and may provide the means necessary the development of control strategies based on the interruption of the gonotrophic cycle of sand flies. Further studies will help towards making such control approaches achievable. Q3
Uncited references Jackson and Locke, 1989 Acknowledgments
Q2
We would like to acknowledge Dr. Isaac Clement for comments lises, of this manuscript and the Núcleo de Microscopia e Microana Universidade Federal de Viçosa, Minas Gerais for technical assistance. Financial support was provided by CAPES and by NIH grant AI074691 to JMR-O. References lez, I.D., Bern, C., Herrero, M., Desjeux, P., Cano, J., Jannin, J., den Boer, M., Alvar, J., Ve Leishmaniasis Control Team, W.H.O., 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7, e35671. Arrese, E.L., Soulages, J.L., 2010. Insect fat body: energy, metabolism, and regulation. Annu Rev. Entomol. 55, 207e225.
9
Bodenstein, D., 1950. The postembryonic development of Drosophila. In: Demerec, M. (Ed.), Biology of Drosophila. Wiley, New York, pp. 275e367. Bryant, B., Raikhel, A.S., 2011. Programmed autophagy in the fat body of Aedes aegypti is required to maintain egg maturation cycles. PloS One 6, e25502. Camargo-Mathias, M.I., Caetano, F.H., 1996. Ultrastructural cytochemistry of oenocytes of Pachycondyla villosa ants (Hymenoptera: Ponerinae). Acta Microsc. 5A, 6000e6003. Cardoso, A.F., Cres, R.L., Moura, A.S., de Almeida, F., Bijovsky, A.T., 2010. Culex quinquefasciatus vitellogenesis: morphological and biochemical aspects. Mem. Inst. Oswaldo Cruz 105, 254e262. Canavoso, L.E., Jouni, Z.E., Karnas, K.J., Pennington, J.E., Wells, M.A., 2001. Fat metabolism in insects. Annu Rev. Nutr. 21, 23e46. Clark, M.K., Dahm, P.A., 1973. Phenobarbital-induced, membrane-like scrolls in the oenocytes of Musca domestica Linnaeus. J. Cell Biol. 56, 870e875. Dean, R.L., Locke, M., Collins, J.V., 1985. Structure of the fat body. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry, and Pharmacology, third ed. Pergamon Press, Oxford, pp. 155e210. DeLoof, A., Lagasse, A., 1970. Juvenile hormone and the ultra structural properties of the fat body of the adult Colorado potato beetle, Leptinotarsa decemlineata Say. Z. Mikrosk. Anat. Forsh. 106, 439e450. Franchini, A., Mandrioli, M., Franceschi, C., Ottaviani, E., 2012. Morpho-functional changes of fat body in bacteria fed Drosophila melanogaster strains. J. Mol. Hist. 43, 243e251. Gutierrez, E., Wiggins, D., Fielding, B., Gould, A.P., 2007. Specialized hepatocyte-like cells regulate Drosophila lipid metabolism. Nature 445, 275e280. Haunerland, N.H., Shirk, P.D., 1995. Regional and functional differentiation in the insect fat body. Annu. Rev. Entomol. 40, 121e145. Haunerland, N.H., Nair, K.K., Bowers, W.S., 1986. A larval specific lipoprotein: purification and characterization of a blue chromoprotein from Heliothis zea. Biochem. Biophys. Res. Commun. 134, 580e586. Haunerland, N.H., Nair, K.K., Bowers, W.S., 1990. Fat body heterogeneity during development of Heliothis zea. Insect Biochem. 20, 829e837. Jackson, A., Locke, M., 1989. The formation of plasma membrane reticular systems in the oenocytes of an insect. Tissue Cell. 21, 463e473. Lauverjat, S., 1977. L'evolution post imaginale due tissue adipeux femelle de Locusta migratoria et son controle endocrine. Gen. Comp. Endocrinol. 33, 13e34. Makki, R., Cinnamon, E., Gould, A.P., 2014. The development and functions of oenocytes. Annu. Rev. Entomol. 9, 405e425. Martins, G.F., Pimenta, P.F.P., 2008. Structural changes in fat body of Aedes aegypti caused by aging and blood feeding. J. Med. Entomol. 45, 1102e1107. Martins, G.F., Serr~ ao, J.E., Ramalho-Ortig~ ao, M., Pimenta, P.F.P., 2011a. A comparative study of fat body morphology in five mosquito species. Mem. Inst. Oswaldo Cruz 106, 742e747. ~o, M., Pimenta, P.F.P., 2011b. Histochemical Martins, G.F., Serr~ ao, J.E., Ramalho-Ortiga and ultrastructural studies of the mosquito Aedes aegypti fat body: effects of aging and diet type. Microsc. Res. Tech. 74, 1032e1039. ~o, J.E., Fortes-Dias, C.L., RamalhoMartins, G.F., Guedes, B.A.M., Silva, L.M., Serra ~o, M., Pimenta, P.F.P., 2011c. Isolation, primary culture and morphological Ortiga characterization of oenocytes from Aedes aegypti pupae. Tissue Cell 43, 83e90. ~o, J.M., 2012. Oenocytes in insects. Invertebr. Surv. J. 9, Martins, G.F., Ramalho-Ortiga 139e152. Raikhel, A.S., Lea, A.O., 1983. Previtellogenic development and synthesis in the fat body of a mosquito: an ultrastructural and imunocytochemical study. Tissue Cell 15, 281e300. Raikhel, A.S., 1986. Lysosomes in the cessation of vitellogenin secretion by the mosquito fat body; selective degradation of Golgi complexes and secretory granules. Tissue Cell 18, 125e142. Raman, C., 2013. Electron microscopy and immunogold labelling analysis of smart ndez-Vilas, A. (Ed.), Current nanoparticles (storage proteins) in insects. In: Me Microscopy Contributions to Advances in Science and Technology. Roma, G.C., Bueno, O.C., Camargo-Mathias, M.I., 2010. Morpho-physiological analysis of the insect fat body: a review. Micron 41, 395e401. Schin, K., Laufer, H., Carr, E., 1977. Cytochemical and electrophoretic studies of haemoglobin synthesis in the fat body of a midge Chironomous thummi. J. Insect Physiol. 23, 1233e1242. Sharma, U., Singh, S., 2008. Insect vectors of Leishmania: distribution, physiology and their control. J. Vector Borne Dis. 45, 255e272. Thomsen, E., 1956. In: Observations on the Oenocytes of the Adult Calliphora erythrocephala. Meig. Bertil Hanstrom. Zool Papers, pp. 298e306. Tobe, S.S., Davey, K.G., 1974. Autoradiographic study of protein synthesis in abdominal tissues of Glossina austeni. Tissue Cell 6, 255e268. Ziegler, R., Ibrahin, M.M., 2001. Formation of lipid reserves in fat body and eggs of the yellow fever mosquito Aedes aegypti. J. Insect Physiol. 47, 623e627.
Please cite this article in press as: Alves de Assis, W., et al., The characterization of the fat bodies and oenocytes in the adult females of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.05.002
61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120