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Pattern of bacteriocyte formation in Periplaneta americana (L.) (Blattaria: Blattidae)
Pergamon Pnnted
PII: SOO20-7322(%)00021-9
PATTERN
OF BACTERIOCYTE FORMATION IN PERIPLANETA (BLATTARIA : BLATTIDAE)
Simonetta *Dipartimento
Lambiase,*
di Biologia
Aldo GrigoloJ
Animale.
Ugo Laudani,
* Luciano
Bntam All rights reserved 0020-73?2!97 S17.00+0.00
AMERICANA
(L.)
Sacchi* and Baccio Baccettit
Universiti di Pavia, Piazza Botta, 9, 27100 Pavia, Italy; and tIstituto Universita di Siena. Via T. Pendola. 62, 53100 Siena, Italy
(Receired
I” Great
di Biologia
Generale,
I2 August 1996; accepted 21 November 1996)
Abstract---In the embryos of Periplaneta americana (L.) (Blattaria : Blattidae), bacterial symbionts, together with vitellophages, form a mycetomic structure inside the deutoplasm; this regresses between the 15th and 16th day after deposition of the ootheca. In this article we describe the migration of bacteria across the wall of the midgut from the mycetome, and the topographic distribution of pre-bacteriocyte cells. We also report that the pre-bacteriocytes are present only on the lateral surface of the internal abdominal fat bodies. We discuss the possible embryological origin and evolution of these cells, and put forward the hypothesis that pre-bacteriocytes are derived from oenocytes activated to perform phagocytosis. 0 1997 Elsevier Science Ltd Index descriptors (in addition haemocytes; oenocytes.
to those in the title): Intracellular
symbiosis;
AND
to whom correspondence
fat body; pre-bacteriocytes;
Analysis of the slides The position of the bacteriocytes was determined on serial sections (355 frontal and 37 sagittal sections). The number of oenocytes and bacteriocytes in the abdominal segments of every frontal and sagittal section was counted. These values from the sections were summed to produce the groups, organized according to thickness, shown in Tables I and 2. Because of the difficulty of determining the boundary of segment VII. the cells of this frontal section were not counted.
RESULTS The intradeutoplasmic mycetome regresses between the 20th and 23rd stage (14-17 days) of embryonic development (Fig. 1). This occurs before the retraction of the vitelline sac from the thorax, its union with the fore- and hind-gut, the closure of the thoracic sclerites and the formation of the bacteriocytes. Moreover, it also marks the final stages of the serosa cell migration into the midgut (our unpublished data). These cells exhibit various degrees of degeneration, which increases towards the cephalic pole. We have also observed these cells in the region where the junction of the midgut with the foregut is located (Fig. 2). The same process then empties the deutoplasmic sac of many vitellophages. In this context, the internal fat bodies are positioned metamerically and show a concave median surface and, adjacent to the hypoderm, a convex lateral surface. On the lateral surface of
METHODS
Insects and their developmental stages Periplaneta americana (L.) (Blattaria : Blattidae) was reared under controlled conditions: 26”C, 65% of relative humidity and a diurnal cycle. The insects were raised on a mixture of fresh lettuce and bread crumbs, enriched with dog food, and received water ad libitum. The ages of the oothecae were estimated from the time of their deposition (age 0). We utilized 1417-day old embryos (developmental stages 2123, after Lenoir-Rousseaux and Lender. 1970).
fAuthor
development;
Histologicul preparations Oothecae were opened in Yeager’s solution (Yeager, 1939) starting from the sutural opening of the keel. Eggs were treated with sodium hypochlorite to obtain dechorionization, then washed twice in Yeager’s solution to interrupt the action of the first step of the treatment. Finally, the vitelline membrane was removed. Embryos were fixed in methanol : acetic acid (3 : I), washed in absolute ethanol for a few minutes and embedded in methacrylate ester. Sections were cut to a thickness of 5 Brn and then stained with Toluidine Blue.
INTRODUCTION During embryonic development in Blattaria, bacterial symbionts migrate from the midgut and penetrate the haemocoel. Here, they are phagocytosed by cells that are then transformed into bacteriocytes (Sacchi et al., 1989). In Blattella germanica, the symbionts cross the ventral wall of the midgut and make their way towards the epineural sinus where Sacchi et al. (1991) observed the formation of bacteriocytes on the ventral fat bodies. However, the precise position of the cells that give rise to the bacteriocytes (here called pre-bacteriocytes) and the dynamics of their development during embryogenesis have never been studied, either in Blattella germanica or in other species of Blattaria. In this work, we describe the topography of the prebacteriocytes and bacteriocytes in the embryos of Periplaneta americana, and discuss a hypothesis for the origin and evolution of pre-bacteriocytes from haemocytes or oenocytes.
MATERIALS
embryonic
should be addressed. 9
et al.
S. Lambiase
10
Table 1. Variation in the number of oenocytes (oe) and bacteriocytes (btc) in the abdominal segments I-VI of a 16-day-old embryo (stage 22) of Periplunefa americana observed in progressive groups of frontal sections, ordered ventro-dorsally. The numbers of cells present in the neural and cardiac sinuses are not estimated. The numbers of oenocytes and bacteriocytes counted within a range from 285 and 1395 pm are shown as an indication of the frequency of the 2 cell types Thickness of the groups of sections (pm) 95* 90 15 50 90 70 95 75 75 70 80 60 70 80 70** *The last dorsal group;
II
I oe
btc
2 2
2 10 12 6
III btc
oe
btc
oe
btc
15 30 53 42 81 13 9 10 21
9 45 51 52 13
I 29 31 26 57 6 5 9 26 9
79 102 89 25 6
5 17 29 20 46 13 25 3 17 33 17 29 85
28 96 122 83 44 1 15
45 95 149 **the first ventral
V
IV
oe
oe
VI btc
oe
btc
2
1 I 13 32 10 23 16 10 19 65 51
10 45 126 20 32 22 28
1 1 1 1 1 13 28 30
6 61 25
172
group
the fat bodies, both dorsally and laterally, is found a monolayer of cells 20-30 nrn in diameter. These cells are rich in pseudopodia and cover an area that varies according to the extension of the internal fat bodies. Files of oenocytes attached to the hypoderm, similar to those described above, are localized at the junction between the sclerites (Fig. 3a, b). The 2 cell types have similar cellular and nuclear diameters, and their chromatin shows similar
Fig. 1. Embryos of Peripluneta americana ( x 200). (a) Typical appearance of the mycetome in a 14-day-old embryo (stage 21); the bacterial mass is evident. (b) A regressed mycetome in a I7-day-old embryo (stage 23) devoid of bacterial symbionts.
degrees of condensation. The cytoplasm of the cells attached to the internal fat bodies is highly vacuolated. These cells are destined to phagocytose the bacterial symbionts, and can, therefore, be considered pre-bacteriocytes. Their distribution is determined by the metameric position of the embryonal fat bodies. Caudally, these cells are positioned latero-ventrally, while towards the cephalic pole they occupy a latero-dorsal position. Pre-bacteriocytes are present in the II-VII abdominal segments. In segments VIII and IX, where the handlage of the hind-gut is found, there are clusters of oenocytes. In this section, the bacteriocytes are never present. The single layer of pre-bacteriocytes rapidly becomes a multilayered cellular mass; this process might result from the active production of oenocytes and their consequent migration to the internal fat bodies (Fig. 3c, d). Haemocytes are present in the abdominal haemocoel, both free in the haemolymph and located intercellularly in the fat bodies. At this stage, one finds neither bacteriocytes nor free bacteria in the haemocoel. The bacterial symbionts migrate from the mycetome without any apparent preferred direction; they are found free in the protoplasmic laciniae, which surround the deutoplasmic masses and adhere to the internal wall of the midgut (Fig. 4). In the course of only a few hours, the situation described above undergoes a profound change: the bacteria, having crossed the wall of the midgut to enter the haemocoel, are phagocytosed by the pre-bacteriocytes that will become bacteriocytes (Fig. 5). As a result, these cells originate the only stem bacteriocyte population from which arise by mitosis, and not as a result of phagocytosis, all other bacteriocytes. At the end of the process of phagocytosis, bacteriocytes (Figs 6 and 7) have the same distribution as that of their pre-bacteriocyte ancestors. Initially, the bacteriocyte complex is V-shaped with arms that curve about the midgut. The “V” opens out towards the cephalic pole
Pattern of Bacteriocyte Formation in Peripluneta americana
and converges posteriorly. The sequence of bacteriocytes along the arms of the “V” is not continuous: they are grouped metamerically. The oenocytes localized along the hypoderm from the fusion zone of the sclerites alternate with the bacteriocytes below (Fig. 8). The variation in the number of oenocytes and bacteriocytes in various abdominal segments is shown in Tables 1 and 2; the spatial distribution of the bacteriocytes is represented graphically in Fig. 9.
DISCUSSION
In both nymphs and adults of some species of Blattaria, bacteriocytes have a characteristic, ordered distribution (Buchner, 1965). In Periplaneta americana, however, bacteriocytes appear to be scattered around the cells of the fat body. In embryos, the distribution of newly formed bacteriocytes has been described in Blattella germanica (Koch, 1933; Sacchi and Grigolo, 1989; Sacchi et al., 1989). Koch identified cells in the fat body with very large nuclei and a homogeneous cytoplasm. He also identified the site-ventrally with respect to the embryonic midgut-of their transformation into bacteriocytes. Initially, the bacteriocytes are clustered around the surface of the adipose lobes adjacent to the intestine, and are later dispersed between the cells of these lobes. Sacchi et al. (1989) identified the system of phagocytosis responsible for the formation of the bacteriocytes, and proposed the hypothesis that pre-bacteriocytes were haemocytes. Sacchi et al. (1988) had already suggested that it was the bacteriocytes, and not the free symbionts, that migrate between the ovarioles. However, they did not analyse the initial distribution of pre-bacteriocytes and embryonic bacteriocytes. In embryos of Periplaneta americana, the early distribution of bacteriocytes, being on the lateral and dorsal surfaces of the fat bodies, is different from that described in Blattella germanica, where it is ventral. The distribution of the embryonic bacteriocytes in the 2 species and that of the symbionts observed in this study beg the question whether the bacteria are able to exit from any part of the gut wall to reach the pre-bacteriocytes, or whether migration is from particular regions of the midgut situated near clusters of pre-bacteriocytes. Another interesting question is raised by the fact that pre-bacteriocytes are found only at particular sites in the fat bodies. If pre-bacteriocytes are thought to be a population of cells differentiated to perform the role of phagocytosis of bacterial symbionts, the lateral surface of the internal abdominal fat bodies would constitute the point of attraction for these cells. If, on the other hand, pre-bacteriocytes are derived from cells not primarily differentiated (oenocytes or haemocytes), the surface of the fat bodies would be the target site and, at the same time, the site of differentiation into pre-bacteriocytes of a part of this population of cells. The hypothesis that pre-bacteriocytes are derived from oenocytes of the abdominal hypoderm would seem ten-
II
12
S. Lambiase
et al.
#.’
rl
Fig. 2. Sixteen-day-old embryos (stage 22). (a) F rontal section showing a mass of degenerating serosa cells at the anterior extremity of the deutoplasmic sac ( x 125). (b) Sagittal section: the integrity of the yolk lamella (yl) is evident ( x 125). (c) A sagittal section in which can be seen degenerating serosa cells into the dorsal haemocoel ( x 125). (d) A close-up ofthe above showing various states of degenerating serosa ( x 500). dw = dorsal wall; h = haemocoel; ps = pericardial sinus; st = stomodeum protruding into the deutoplasmic sac.
Pattern
of Bacteriocyte
Formation
in Periplaneta americana
Fig. 3. Frontal sections of Periplaneta americana embryos. (a) Fourteen-day-old embryo (stage 21) showing the monolayer of prebacteriocytes (arrows) on the internal abdominal fat body ( x 125). (b) At higher magnification ( x 500) the pseudopodia of the prebacteriocytes are evident (arrows). (c) In a 15-day-old embryo (stage 21) masses of pre-bacteriocytes (arrows) cover the lateral part of the internal fat bodies (x 125). (d) Close-up of the above: a central mass of pre-bacteriocytes exhibiting numerous pseudopodia. Oenocytes (arrows), which also possess pseudopodia, are positioned peripherally ( x 500). d = deutoplasm; he = haemocytes: my = myoblasts; oe = oenocytes; p = proctodeum.
13
S. Lambiase et al.
Fig. 4. Sagittal sections of a 16-day-old embryo (stage 22) ( x 500). (a) Bacteria (arrows) leaving the mycetome (m). (b) A mass of symbionts located near to the ventral portion of the yolk lamella (~1).(c) Masses of symbionts in the dorsal portion of the yolk lamella (~1).d = deutoplasm; p = proctodeum; v = vitellophages; vw = ventral wall.
Pattern
of Bacteriocyte
Formation
in Periphneta
americanu
Fig. 5. Sagittal section of a 16-day-old embryo (stage 22) showing the beginning of the process of phagocytosis by the pre-bacteriocytes ( x 500).
is completed. Externally. the dorsal Fig. 6. Sagittal section of a 16-day-old embryo (stage 22) (x 12). The process of phagocytosis (arrl ows) and lateral (arrowheads) distribution of bacteriocytes on the surface of the internal fat bodies is evidenced. I, II and III, first, second and third abdominal segments; oe = oenocytes.
16
S. Lambiase et al.
Fig. 7. Serial sections of a 16-day-o Id embryo (stage 22) showing the caudo-cephalic localization of bacteriocytes ( x 5). In the ventral sectic m (a), the bat teriocytes (arrows) are localized only in the terminal part of the embryo. In section b, intermediate bet a and c, the bacteriocytes (arrows) are localized towards the central abdominal segments, and in the most dorsal section (c) they (arrows) occupy the first abdominal segments. h=heart.
Pattern of Bacteriocyte Formation in
Periplaneta
17
americana
Fig. 8. Reconstructed frontal section of a l6-day-old embryo (stage 22) (x 15). Four segments, 2 of which complete, are shown. Junctions are indicated by bold arrows. Within each segment, the alternation of oenocytes (arrowheads) and bacteriocytes (light arrows) attached to the internal fat bodies is evident. d = deutoplasm.
DORSAL
7
median plane
VENTRAL Fig. 9. A three-dimensional representation of bacteriocyte distribution (grey boxes) in a 16-day-old embryo (stage 22) of Periplanera americana. The diagram was constructed by integrating the data from the frontal (Table 1) and sagittal (Table 2) sections.
18
S. Lambiase et al.
able on the grounds that morphologically they are very similar. Additionally, these oenocytes are capable of migration (Kramer and Wigglesworth, 1950) and we observed in them the presence of pseudopodia only before the formation of bacteriocytes. Pre-bacteriocytes, therefore, could be considered oenocytes, which have moved towards the internal fat bodies and then become activated for phagocytosis. That pre-bacteriocytes are derived from haemocytes, however, is supported by the work of Anderson et al. (1973) who observed their phagocytic activity in vitro (see also Gupta, 1986). Although we did not observe morphological similarity between haemocytes in haemolymph and pre-bacteriocytes, we cannot exclude from consideration the possibility that pre-bacteriocytes are derived from haemocytes transformed by interaction with the fat bodies. If it is correct that hypodermal oenocytes or haemocytes are stimulated to transform themselves into prebacteriocytes, there must be an interaction between these cells and the target area of the fat bodies. In fact, the competence for phagocytosis of symbionts is not evidenced in hypodermal oenocytes and in free haemocytes. The critical point of this hypothesis is the fact that, although the fat bodies, haemocytes, and oenocytes are found throughout the embryo, the activation of pre-bacteriocytes from haemocytes/oenocytes occurs only in specific regions of the abdominal fat bodies. The hypothesis that there exists a cell lineage that is specialized for the phagocytosis of bacterial symbionts is supported by the observations of Buchner (1918), Buchner (1921) Hecht (1924) Buchner (1928) and Kolb (1957). These workers compared species of ants that possess endocellular bacteria with those that do not. In the latter are found, in the place of bacteriocytes, cells with a very characteristic morphology. This was interpreted to indicate that the bacteriocytes had lost their bacteria during the course of evolution. Mansour (1927, 1930) reported, in an aposymbiotic variety of Calandra oryzae, the presence of morphologically distinctive cells, which he interpreted to be “sterile bacteriocytes”. These were shown to occupy sites in which, in varieties containing symbionts, one finds normal bacteriocytes. Furthermore, in aposymbiotic embryos of Blattella germanica, raised from experimentally aposymbiotic females, one finds clusters of cells devoid of bacteria (“empty bacteriocytes”), distributed in II-VI abdominal segments, precisely in those positions where, in the normal embryos, the bacteriocytes are found (Brooks and Richards, 1955). These cells do not undergo mitosis and, even in the absence of bacterial infection, can be considered part of a lineage evolutionarily differentiated for endosymbiosis. The present knowledge on the basis of the acquired data does not allow us to consider the hypothesis of the evolutionary differentiation in insects more reliable than that of the transformation of cellular (oenocytic or haemocytic) lineages, which are primarily adapted for other functions.
In support of the hypothesis that pre-bacteriocytes can arise from oenocytes, there is evidence that in some species of Diptera, Nematocera, the oenocytes harbour Microsporida (Becnel et al., 1986; Sweeney et al., 1990; Andreadis, 1994) and, in some cases, also transfer them to the ovarian cells (Andreadis and Hall, 1979; Dickson and Barr, 1990). This latter function is comparable to that normally performed in the transmission of bacteria in Periplaneta americana. Therefore, we cannot exclude that in Blattaria the oenocytes, besides being the source of ecdysone and the stimulus for molting (Locke, 1969) may also harbour bacterial symbionts. Acknowledgements-We
are grateful to A. Tronconi for his technical assistance. The work was supported by the Minister0 dell’Universit8 e della Ricerca Scientifica e Tecnologica, MURST.
REFERENCES Anderson, R. S., B. Holmes. and R. A. Good. 1973. In vitro bactericidal capacity of Blaberus craniifer hemocytes. J. Invertebr. Pathol. 22: 127-135.
Andreadis, T. G. 1994. Host range tests with Edhazardia aedis (Microsporida : Culicosporidae) against northern Neoartic mosquitoes. J Invertebr. Pathol. 64: 4651.
Andreadis, T. G. and D. W Hall. 1979. Development, ultrastructure, and mode of transmission of Amblyospora sp. (Microspora) in the mosquito. J. Protozool. 26: 444452. Becnel, J. J., E. I. Hazard. and T. Fukuda. 1986. Fine structure and development of Pilosporella chapmani (Microspora : Thelohaniidae) in the mosquito Aedes triseriatus (Say). J. Protozool. 33: 60-66. Brooks, M. A. and A. G. Richards. 1955. Intracellular symbiosis in cockroaches. II. Mitotic division of mycetocytes. Science (Wash., DC) 122: 242. Buchner, P. 1918. Die akzessorischen Kerne des Hymenoptereneies. Arch. Mikrosk. Anat. EntwMech. 91: l-212. Buchner, P. 1921. Tier und Pjlanze in intrazellularer Symbiose. Borntraeger, Berlin. Buchner, P. 1928. Die Grenzen des Symbioseprinzipes. Naturwissenschaften 16: 5 17-52 1. Buchner, P. 1965. Endosymbiosis of Animals with Plant Microorganisms. Interscience, New York. Dickson, D. L. and A. R. Barr. 1990. Development of Amblyospora campbelli (Microsporida : Amblyosporidae) in the mosquito Culiseta incidens (Thomson). J. Protozool. 37: 71-78. Gupta, A.P. 1986. Arthropod immunocytes. Identification, structure, functions, and analogies to the functions of vertebrate B- and Tlymphocytes. pp. 3-59. In A.P. Gupta (ed). Hemocytic and Humoral Immunity in Arthropods. Wiley, New York. Hecht, 0. 1924. Embryonalentwicklung und Symbiose bei Camponotus ligniperda. Z. Wiss. 2001. 122: 173-204. Koch, A. 1933. Uber Kiinstlich symbiontenfrei gemachte Insekten. Verh. Deutsch Zool. Ges. 35: 1433150.
Kolb, G. 1957. Uber die Kernverhaltnisse der Symbionten von Camponotus ligniperda Latr., Aphrophora salicis de G. und Pediculus vestimenti Burm. Naturwissenschaften 44: 546. Kramer, S. and V. B. Wigglesworth. 1950. The outer layers of the cuticle in the cockroach Periplaneta americana and the function of the oenocytes. Quart. J. Microsc. Sci. 91: 62-73. Lenoir-Rousseaux, J. J. and T. Lender. 1970. Table de developpement embryonnaire de Periplaneta americana (L.) Insecte. Dictyoptere. Bull. Sot. Zool. Fr. 95: 737-75 1. Locke, M. 1969. The ultrastructure of the oenocytes in the molt/ intermolt cycle of an insect. Tissue Cell 1: 103-l 54. Mansour, K. 1927. The development of the larval and adult mid-gut of Calandra oryzae (L.): the rice weevil. Quart. J. Microsc. Sci. 71: 313-352.
Mansour, K. 1930. Preliminary studies on the bacterial cell-mass (accessory cell-mass) of Calandra oryzae (L.): the rice weevil. Quart. J. Microsc. Sci. 73: 421436.
Pattern
of Bacteriocyte
Formation
Sacchi, L. and A. Grigolo. 1989. Endocytobiosis in Blattellagermanica: recent acquisitions. Endocyt. Cell Res. 6: 121-147. Sacchi, L., P. De Piceis Poker, A. Grigolo, E. Bigliardi, M. G. Selmi, B. Baccetti. and U. Laudani. 1989. Origin of bacteriocytes in Bluttellu germanica L. Endocytobiology 4: 389-391. Sacchi, L., A. Grigolo, A., P. De Piceis Poker, E. Bigliardi. and U. Laudani. 1991. Endocytobiosis in Dictyoptera: the transmission of symbiotic bacteria in Blattella germanica L. (Dictyoptera Blattellidae). Ethel. Ecol. E~ol. 1: 155-158. Sacchi, L.. A. Grigolo, M. Mazzini, E. Bigliardi, B. Baccetti. and U.
in Peripluneta americanu
19
Laudani. 1988. Symbionts in the oocytes of Bluttellagermanica (L.) (Dictyoptera : Blattellidae): their mode of transmission. Znt. J. Insect Morphol. Embryol. 17: 437446. Sweeney, A. W., S. L. Doggett. and R. G. Piper. 1990. Life cycle of Amblyospora indicola (Microspora : Amblyosporidae), a parasit of the mosquito Culex sitiens and of Apoqxlops sp. Copepods. J. Invertebr. Pathol. 55: 428434. Yeager, J. F. 1939. Electrical stimulation of isolated heart of Peripluneta americana. J. Agric. Res. 59: 121-138.
PII: SOO20-7322(%)00021-9
PATTERN
OF BACTERIOCYTE FORMATION IN PERIPLANETA (BLATTARIA : BLATTIDAE)
Simonetta *Dipartimento
Lambiase,*
di Biologia
Aldo GrigoloJ
Animale.
Ugo Laudani,
* Luciano
Bntam All rights reserved 0020-73?2!97 S17.00+0.00
AMERICANA
(L.)
Sacchi* and Baccio Baccettit
Universiti di Pavia, Piazza Botta, 9, 27100 Pavia, Italy; and tIstituto Universita di Siena. Via T. Pendola. 62, 53100 Siena, Italy
(Receired
I” Great
di Biologia
Generale,
I2 August 1996; accepted 21 November 1996)
Abstract---In the embryos of Periplaneta americana (L.) (Blattaria : Blattidae), bacterial symbionts, together with vitellophages, form a mycetomic structure inside the deutoplasm; this regresses between the 15th and 16th day after deposition of the ootheca. In this article we describe the migration of bacteria across the wall of the midgut from the mycetome, and the topographic distribution of pre-bacteriocyte cells. We also report that the pre-bacteriocytes are present only on the lateral surface of the internal abdominal fat bodies. We discuss the possible embryological origin and evolution of these cells, and put forward the hypothesis that pre-bacteriocytes are derived from oenocytes activated to perform phagocytosis. 0 1997 Elsevier Science Ltd Index descriptors (in addition haemocytes; oenocytes.
to those in the title): Intracellular
symbiosis;
AND
to whom correspondence
fat body; pre-bacteriocytes;
Analysis of the slides The position of the bacteriocytes was determined on serial sections (355 frontal and 37 sagittal sections). The number of oenocytes and bacteriocytes in the abdominal segments of every frontal and sagittal section was counted. These values from the sections were summed to produce the groups, organized according to thickness, shown in Tables I and 2. Because of the difficulty of determining the boundary of segment VII. the cells of this frontal section were not counted.
RESULTS The intradeutoplasmic mycetome regresses between the 20th and 23rd stage (14-17 days) of embryonic development (Fig. 1). This occurs before the retraction of the vitelline sac from the thorax, its union with the fore- and hind-gut, the closure of the thoracic sclerites and the formation of the bacteriocytes. Moreover, it also marks the final stages of the serosa cell migration into the midgut (our unpublished data). These cells exhibit various degrees of degeneration, which increases towards the cephalic pole. We have also observed these cells in the region where the junction of the midgut with the foregut is located (Fig. 2). The same process then empties the deutoplasmic sac of many vitellophages. In this context, the internal fat bodies are positioned metamerically and show a concave median surface and, adjacent to the hypoderm, a convex lateral surface. On the lateral surface of
METHODS
Insects and their developmental stages Periplaneta americana (L.) (Blattaria : Blattidae) was reared under controlled conditions: 26”C, 65% of relative humidity and a diurnal cycle. The insects were raised on a mixture of fresh lettuce and bread crumbs, enriched with dog food, and received water ad libitum. The ages of the oothecae were estimated from the time of their deposition (age 0). We utilized 1417-day old embryos (developmental stages 2123, after Lenoir-Rousseaux and Lender. 1970).
fAuthor
development;
Histologicul preparations Oothecae were opened in Yeager’s solution (Yeager, 1939) starting from the sutural opening of the keel. Eggs were treated with sodium hypochlorite to obtain dechorionization, then washed twice in Yeager’s solution to interrupt the action of the first step of the treatment. Finally, the vitelline membrane was removed. Embryos were fixed in methanol : acetic acid (3 : I), washed in absolute ethanol for a few minutes and embedded in methacrylate ester. Sections were cut to a thickness of 5 Brn and then stained with Toluidine Blue.
INTRODUCTION During embryonic development in Blattaria, bacterial symbionts migrate from the midgut and penetrate the haemocoel. Here, they are phagocytosed by cells that are then transformed into bacteriocytes (Sacchi et al., 1989). In Blattella germanica, the symbionts cross the ventral wall of the midgut and make their way towards the epineural sinus where Sacchi et al. (1991) observed the formation of bacteriocytes on the ventral fat bodies. However, the precise position of the cells that give rise to the bacteriocytes (here called pre-bacteriocytes) and the dynamics of their development during embryogenesis have never been studied, either in Blattella germanica or in other species of Blattaria. In this work, we describe the topography of the prebacteriocytes and bacteriocytes in the embryos of Periplaneta americana, and discuss a hypothesis for the origin and evolution of pre-bacteriocytes from haemocytes or oenocytes.
MATERIALS
embryonic
should be addressed. 9
et al.
S. Lambiase
10
Table 1. Variation in the number of oenocytes (oe) and bacteriocytes (btc) in the abdominal segments I-VI of a 16-day-old embryo (stage 22) of Periplunefa americana observed in progressive groups of frontal sections, ordered ventro-dorsally. The numbers of cells present in the neural and cardiac sinuses are not estimated. The numbers of oenocytes and bacteriocytes counted within a range from 285 and 1395 pm are shown as an indication of the frequency of the 2 cell types Thickness of the groups of sections (pm) 95* 90 15 50 90 70 95 75 75 70 80 60 70 80 70** *The last dorsal group;
II
I oe
btc
2 2
2 10 12 6
III btc
oe
btc
oe
btc
15 30 53 42 81 13 9 10 21
9 45 51 52 13
I 29 31 26 57 6 5 9 26 9
79 102 89 25 6
5 17 29 20 46 13 25 3 17 33 17 29 85
28 96 122 83 44 1 15
45 95 149 **the first ventral
V
IV
oe
oe
VI btc
oe
btc
2
1 I 13 32 10 23 16 10 19 65 51
10 45 126 20 32 22 28
1 1 1 1 1 13 28 30
6 61 25
172
group
the fat bodies, both dorsally and laterally, is found a monolayer of cells 20-30 nrn in diameter. These cells are rich in pseudopodia and cover an area that varies according to the extension of the internal fat bodies. Files of oenocytes attached to the hypoderm, similar to those described above, are localized at the junction between the sclerites (Fig. 3a, b). The 2 cell types have similar cellular and nuclear diameters, and their chromatin shows similar
Fig. 1. Embryos of Peripluneta americana ( x 200). (a) Typical appearance of the mycetome in a 14-day-old embryo (stage 21); the bacterial mass is evident. (b) A regressed mycetome in a I7-day-old embryo (stage 23) devoid of bacterial symbionts.
degrees of condensation. The cytoplasm of the cells attached to the internal fat bodies is highly vacuolated. These cells are destined to phagocytose the bacterial symbionts, and can, therefore, be considered pre-bacteriocytes. Their distribution is determined by the metameric position of the embryonal fat bodies. Caudally, these cells are positioned latero-ventrally, while towards the cephalic pole they occupy a latero-dorsal position. Pre-bacteriocytes are present in the II-VII abdominal segments. In segments VIII and IX, where the handlage of the hind-gut is found, there are clusters of oenocytes. In this section, the bacteriocytes are never present. The single layer of pre-bacteriocytes rapidly becomes a multilayered cellular mass; this process might result from the active production of oenocytes and their consequent migration to the internal fat bodies (Fig. 3c, d). Haemocytes are present in the abdominal haemocoel, both free in the haemolymph and located intercellularly in the fat bodies. At this stage, one finds neither bacteriocytes nor free bacteria in the haemocoel. The bacterial symbionts migrate from the mycetome without any apparent preferred direction; they are found free in the protoplasmic laciniae, which surround the deutoplasmic masses and adhere to the internal wall of the midgut (Fig. 4). In the course of only a few hours, the situation described above undergoes a profound change: the bacteria, having crossed the wall of the midgut to enter the haemocoel, are phagocytosed by the pre-bacteriocytes that will become bacteriocytes (Fig. 5). As a result, these cells originate the only stem bacteriocyte population from which arise by mitosis, and not as a result of phagocytosis, all other bacteriocytes. At the end of the process of phagocytosis, bacteriocytes (Figs 6 and 7) have the same distribution as that of their pre-bacteriocyte ancestors. Initially, the bacteriocyte complex is V-shaped with arms that curve about the midgut. The “V” opens out towards the cephalic pole
Pattern of Bacteriocyte Formation in Peripluneta americana
and converges posteriorly. The sequence of bacteriocytes along the arms of the “V” is not continuous: they are grouped metamerically. The oenocytes localized along the hypoderm from the fusion zone of the sclerites alternate with the bacteriocytes below (Fig. 8). The variation in the number of oenocytes and bacteriocytes in various abdominal segments is shown in Tables 1 and 2; the spatial distribution of the bacteriocytes is represented graphically in Fig. 9.
DISCUSSION
In both nymphs and adults of some species of Blattaria, bacteriocytes have a characteristic, ordered distribution (Buchner, 1965). In Periplaneta americana, however, bacteriocytes appear to be scattered around the cells of the fat body. In embryos, the distribution of newly formed bacteriocytes has been described in Blattella germanica (Koch, 1933; Sacchi and Grigolo, 1989; Sacchi et al., 1989). Koch identified cells in the fat body with very large nuclei and a homogeneous cytoplasm. He also identified the site-ventrally with respect to the embryonic midgut-of their transformation into bacteriocytes. Initially, the bacteriocytes are clustered around the surface of the adipose lobes adjacent to the intestine, and are later dispersed between the cells of these lobes. Sacchi et al. (1989) identified the system of phagocytosis responsible for the formation of the bacteriocytes, and proposed the hypothesis that pre-bacteriocytes were haemocytes. Sacchi et al. (1988) had already suggested that it was the bacteriocytes, and not the free symbionts, that migrate between the ovarioles. However, they did not analyse the initial distribution of pre-bacteriocytes and embryonic bacteriocytes. In embryos of Periplaneta americana, the early distribution of bacteriocytes, being on the lateral and dorsal surfaces of the fat bodies, is different from that described in Blattella germanica, where it is ventral. The distribution of the embryonic bacteriocytes in the 2 species and that of the symbionts observed in this study beg the question whether the bacteria are able to exit from any part of the gut wall to reach the pre-bacteriocytes, or whether migration is from particular regions of the midgut situated near clusters of pre-bacteriocytes. Another interesting question is raised by the fact that pre-bacteriocytes are found only at particular sites in the fat bodies. If pre-bacteriocytes are thought to be a population of cells differentiated to perform the role of phagocytosis of bacterial symbionts, the lateral surface of the internal abdominal fat bodies would constitute the point of attraction for these cells. If, on the other hand, pre-bacteriocytes are derived from cells not primarily differentiated (oenocytes or haemocytes), the surface of the fat bodies would be the target site and, at the same time, the site of differentiation into pre-bacteriocytes of a part of this population of cells. The hypothesis that pre-bacteriocytes are derived from oenocytes of the abdominal hypoderm would seem ten-
II
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et al.
#.’
rl
Fig. 2. Sixteen-day-old embryos (stage 22). (a) F rontal section showing a mass of degenerating serosa cells at the anterior extremity of the deutoplasmic sac ( x 125). (b) Sagittal section: the integrity of the yolk lamella (yl) is evident ( x 125). (c) A sagittal section in which can be seen degenerating serosa cells into the dorsal haemocoel ( x 125). (d) A close-up ofthe above showing various states of degenerating serosa ( x 500). dw = dorsal wall; h = haemocoel; ps = pericardial sinus; st = stomodeum protruding into the deutoplasmic sac.
Pattern
of Bacteriocyte
Formation
in Periplaneta americana
Fig. 3. Frontal sections of Periplaneta americana embryos. (a) Fourteen-day-old embryo (stage 21) showing the monolayer of prebacteriocytes (arrows) on the internal abdominal fat body ( x 125). (b) At higher magnification ( x 500) the pseudopodia of the prebacteriocytes are evident (arrows). (c) In a 15-day-old embryo (stage 21) masses of pre-bacteriocytes (arrows) cover the lateral part of the internal fat bodies (x 125). (d) Close-up of the above: a central mass of pre-bacteriocytes exhibiting numerous pseudopodia. Oenocytes (arrows), which also possess pseudopodia, are positioned peripherally ( x 500). d = deutoplasm; he = haemocytes: my = myoblasts; oe = oenocytes; p = proctodeum.
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Fig. 4. Sagittal sections of a 16-day-old embryo (stage 22) ( x 500). (a) Bacteria (arrows) leaving the mycetome (m). (b) A mass of symbionts located near to the ventral portion of the yolk lamella (~1).(c) Masses of symbionts in the dorsal portion of the yolk lamella (~1).d = deutoplasm; p = proctodeum; v = vitellophages; vw = ventral wall.
Pattern
of Bacteriocyte
Formation
in Periphneta
americanu
Fig. 5. Sagittal section of a 16-day-old embryo (stage 22) showing the beginning of the process of phagocytosis by the pre-bacteriocytes ( x 500).
is completed. Externally. the dorsal Fig. 6. Sagittal section of a 16-day-old embryo (stage 22) (x 12). The process of phagocytosis (arrl ows) and lateral (arrowheads) distribution of bacteriocytes on the surface of the internal fat bodies is evidenced. I, II and III, first, second and third abdominal segments; oe = oenocytes.
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Fig. 7. Serial sections of a 16-day-o Id embryo (stage 22) showing the caudo-cephalic localization of bacteriocytes ( x 5). In the ventral sectic m (a), the bat teriocytes (arrows) are localized only in the terminal part of the embryo. In section b, intermediate bet a and c, the bacteriocytes (arrows) are localized towards the central abdominal segments, and in the most dorsal section (c) they (arrows) occupy the first abdominal segments. h=heart.
Pattern of Bacteriocyte Formation in
Periplaneta
17
americana
Fig. 8. Reconstructed frontal section of a l6-day-old embryo (stage 22) (x 15). Four segments, 2 of which complete, are shown. Junctions are indicated by bold arrows. Within each segment, the alternation of oenocytes (arrowheads) and bacteriocytes (light arrows) attached to the internal fat bodies is evident. d = deutoplasm.
DORSAL
7
median plane
VENTRAL Fig. 9. A three-dimensional representation of bacteriocyte distribution (grey boxes) in a 16-day-old embryo (stage 22) of Periplanera americana. The diagram was constructed by integrating the data from the frontal (Table 1) and sagittal (Table 2) sections.
18
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able on the grounds that morphologically they are very similar. Additionally, these oenocytes are capable of migration (Kramer and Wigglesworth, 1950) and we observed in them the presence of pseudopodia only before the formation of bacteriocytes. Pre-bacteriocytes, therefore, could be considered oenocytes, which have moved towards the internal fat bodies and then become activated for phagocytosis. That pre-bacteriocytes are derived from haemocytes, however, is supported by the work of Anderson et al. (1973) who observed their phagocytic activity in vitro (see also Gupta, 1986). Although we did not observe morphological similarity between haemocytes in haemolymph and pre-bacteriocytes, we cannot exclude from consideration the possibility that pre-bacteriocytes are derived from haemocytes transformed by interaction with the fat bodies. If it is correct that hypodermal oenocytes or haemocytes are stimulated to transform themselves into prebacteriocytes, there must be an interaction between these cells and the target area of the fat bodies. In fact, the competence for phagocytosis of symbionts is not evidenced in hypodermal oenocytes and in free haemocytes. The critical point of this hypothesis is the fact that, although the fat bodies, haemocytes, and oenocytes are found throughout the embryo, the activation of pre-bacteriocytes from haemocytes/oenocytes occurs only in specific regions of the abdominal fat bodies. The hypothesis that there exists a cell lineage that is specialized for the phagocytosis of bacterial symbionts is supported by the observations of Buchner (1918), Buchner (1921) Hecht (1924) Buchner (1928) and Kolb (1957). These workers compared species of ants that possess endocellular bacteria with those that do not. In the latter are found, in the place of bacteriocytes, cells with a very characteristic morphology. This was interpreted to indicate that the bacteriocytes had lost their bacteria during the course of evolution. Mansour (1927, 1930) reported, in an aposymbiotic variety of Calandra oryzae, the presence of morphologically distinctive cells, which he interpreted to be “sterile bacteriocytes”. These were shown to occupy sites in which, in varieties containing symbionts, one finds normal bacteriocytes. Furthermore, in aposymbiotic embryos of Blattella germanica, raised from experimentally aposymbiotic females, one finds clusters of cells devoid of bacteria (“empty bacteriocytes”), distributed in II-VI abdominal segments, precisely in those positions where, in the normal embryos, the bacteriocytes are found (Brooks and Richards, 1955). These cells do not undergo mitosis and, even in the absence of bacterial infection, can be considered part of a lineage evolutionarily differentiated for endosymbiosis. The present knowledge on the basis of the acquired data does not allow us to consider the hypothesis of the evolutionary differentiation in insects more reliable than that of the transformation of cellular (oenocytic or haemocytic) lineages, which are primarily adapted for other functions.
In support of the hypothesis that pre-bacteriocytes can arise from oenocytes, there is evidence that in some species of Diptera, Nematocera, the oenocytes harbour Microsporida (Becnel et al., 1986; Sweeney et al., 1990; Andreadis, 1994) and, in some cases, also transfer them to the ovarian cells (Andreadis and Hall, 1979; Dickson and Barr, 1990). This latter function is comparable to that normally performed in the transmission of bacteria in Periplaneta americana. Therefore, we cannot exclude that in Blattaria the oenocytes, besides being the source of ecdysone and the stimulus for molting (Locke, 1969) may also harbour bacterial symbionts. Acknowledgements-We
are grateful to A. Tronconi for his technical assistance. The work was supported by the Minister0 dell’Universit8 e della Ricerca Scientifica e Tecnologica, MURST.
REFERENCES Anderson, R. S., B. Holmes. and R. A. Good. 1973. In vitro bactericidal capacity of Blaberus craniifer hemocytes. J. Invertebr. Pathol. 22: 127-135.
Andreadis, T. G. 1994. Host range tests with Edhazardia aedis (Microsporida : Culicosporidae) against northern Neoartic mosquitoes. J Invertebr. Pathol. 64: 4651.
Andreadis, T. G. and D. W Hall. 1979. Development, ultrastructure, and mode of transmission of Amblyospora sp. (Microspora) in the mosquito. J. Protozool. 26: 444452. Becnel, J. J., E. I. Hazard. and T. Fukuda. 1986. Fine structure and development of Pilosporella chapmani (Microspora : Thelohaniidae) in the mosquito Aedes triseriatus (Say). J. Protozool. 33: 60-66. Brooks, M. A. and A. G. Richards. 1955. Intracellular symbiosis in cockroaches. II. Mitotic division of mycetocytes. Science (Wash., DC) 122: 242. Buchner, P. 1918. Die akzessorischen Kerne des Hymenoptereneies. Arch. Mikrosk. Anat. EntwMech. 91: l-212. Buchner, P. 1921. Tier und Pjlanze in intrazellularer Symbiose. Borntraeger, Berlin. Buchner, P. 1928. Die Grenzen des Symbioseprinzipes. Naturwissenschaften 16: 5 17-52 1. Buchner, P. 1965. Endosymbiosis of Animals with Plant Microorganisms. Interscience, New York. Dickson, D. L. and A. R. Barr. 1990. Development of Amblyospora campbelli (Microsporida : Amblyosporidae) in the mosquito Culiseta incidens (Thomson). J. Protozool. 37: 71-78. Gupta, A.P. 1986. Arthropod immunocytes. Identification, structure, functions, and analogies to the functions of vertebrate B- and Tlymphocytes. pp. 3-59. In A.P. Gupta (ed). Hemocytic and Humoral Immunity in Arthropods. Wiley, New York. Hecht, 0. 1924. Embryonalentwicklung und Symbiose bei Camponotus ligniperda. Z. Wiss. 2001. 122: 173-204. Koch, A. 1933. Uber Kiinstlich symbiontenfrei gemachte Insekten. Verh. Deutsch Zool. Ges. 35: 1433150.
Kolb, G. 1957. Uber die Kernverhaltnisse der Symbionten von Camponotus ligniperda Latr., Aphrophora salicis de G. und Pediculus vestimenti Burm. Naturwissenschaften 44: 546. Kramer, S. and V. B. Wigglesworth. 1950. The outer layers of the cuticle in the cockroach Periplaneta americana and the function of the oenocytes. Quart. J. Microsc. Sci. 91: 62-73. Lenoir-Rousseaux, J. J. and T. Lender. 1970. Table de developpement embryonnaire de Periplaneta americana (L.) Insecte. Dictyoptere. Bull. Sot. Zool. Fr. 95: 737-75 1. Locke, M. 1969. The ultrastructure of the oenocytes in the molt/ intermolt cycle of an insect. Tissue Cell 1: 103-l 54. Mansour, K. 1927. The development of the larval and adult mid-gut of Calandra oryzae (L.): the rice weevil. Quart. J. Microsc. Sci. 71: 313-352.
Mansour, K. 1930. Preliminary studies on the bacterial cell-mass (accessory cell-mass) of Calandra oryzae (L.): the rice weevil. Quart. J. Microsc. Sci. 73: 421436.
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of Bacteriocyte
Formation
Sacchi, L. and A. Grigolo. 1989. Endocytobiosis in Blattellagermanica: recent acquisitions. Endocyt. Cell Res. 6: 121-147. Sacchi, L., P. De Piceis Poker, A. Grigolo, E. Bigliardi, M. G. Selmi, B. Baccetti. and U. Laudani. 1989. Origin of bacteriocytes in Bluttellu germanica L. Endocytobiology 4: 389-391. Sacchi, L., A. Grigolo, A., P. De Piceis Poker, E. Bigliardi. and U. Laudani. 1991. Endocytobiosis in Dictyoptera: the transmission of symbiotic bacteria in Blattella germanica L. (Dictyoptera Blattellidae). Ethel. Ecol. E~ol. 1: 155-158. Sacchi, L.. A. Grigolo, M. Mazzini, E. Bigliardi, B. Baccetti. and U.
in Peripluneta americanu
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Laudani. 1988. Symbionts in the oocytes of Bluttellagermanica (L.) (Dictyoptera : Blattellidae): their mode of transmission. Znt. J. Insect Morphol. Embryol. 17: 437446. Sweeney, A. W., S. L. Doggett. and R. G. Piper. 1990. Life cycle of Amblyospora indicola (Microspora : Amblyosporidae), a parasit of the mosquito Culex sitiens and of Apoqxlops sp. Copepods. J. Invertebr. Pathol. 55: 428434. Yeager, J. F. 1939. Electrical stimulation of isolated heart of Peripluneta americana. J. Agric. Res. 59: 121-138.