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Morphological Study of the Larval Spiracular System in Eight Lutzomyia Species (Diptera: Psychodidae) Anna Maria Fausto, M Dora Feliciangeli*^/+, Michele Maroli**, Massimo Mazzini
Dipartimento di Scienze Ambientali, Università della Tuscia, Viterbo,
Italy Received 20 June 1997; Accepted 27 August 1997 Code Number:OC98014 Sizes of Files: Text: 26.5K Graphics: Line drawings and photographs (jpg) - 191.8K Tables (jpg) - 31.9K
The morphology of the spiracles of fourth instar larva in eight sandfly species were examined by light and scanning electron microscopy. Species studied were: Lutzomyia longipalpis (Lutz & Neiva), L. ovallesi (Ortiz), L. youngi Feliciangeli & Murillo, L. evansi (Nunez-Tovar), L. trinidadensis (Newstead), L. migonei (Franca), L. absonodonta Feliciangeli, and L. venezuelensis (Floch & Abonnenc). In larvae of all eight species both thoracic and abdominal spiracles are located at the top of a globular bulge. Their structure consists of a spiracular plate with a sclerotized central portion and a rose-like peripheral portion. The latter has circularly arranged papillae, separated from each other by elongated septa. Each papilla is longitudinally crossed by a fine cleft dividing it into two identical parts. The taxonomic and adaptative value of spiracular morphology is discussed. Key words: sandfly - thoracic and abdominal spiracles - amphipneustic larvae - light and scanning electron microscopy In Insecta, the larval spiracular system assumes a great variety of forms, many of which are clearly adaptative. Despite such as indicative signal, little work has been carried out on the spiracular system in larval stages of different insects (Beckel 1958, Hinton 1967, Berberet & Helms 1972, Khole 1979, Roberts 1981, Nikam & Khole 1989, Principato & Tosti, 1988, 1989). It is generally accepted that the highest number of functional spiracles in existing insects is ten pairs, two of which are thoracic and eight abdominal. On the basis of the number and location of functional spiracles, Keilin (1944) proposed a classification of the larval spiracular system that has been followed until now. In Diptera, the internal morphology of the tracheal system is rather constant in the various families, while numerous variations are evident in spiracles (Whitten 1955). The structure of spiracles in dipterous larvae varies not only with the species but often with their position on the body (Keilin 1944). Thus, each species may show different kinds of spiracles. Such polymorphism is often intimately connected with the respiratory adaptation of insect larval stages to surrounding conditions. The study of the respiratory adaptations of dipterous larvae to the great variety of external factors revealed forms of convergency of certain structures in species phylogenetically distinct as well as divergency in closely allied groups (Keilin 1944, Whitten 1955). For only a few of the species of phlebotomine sandflies were the morphological characters of immature stages been studied in the past (Grassi 1907, Saccà 1950, Abonnenc 1956, Abonnenc & Larivière 1957, Trouillet 1976, 1977, 1979). With the current increase in the colonization and rearing of many sandfly species (Killick-Kendrick et al. 1991), there are now opportunities to describe the morphology of eggs, larvae and pupae of the colonized species (Lane & El Sawaf 1986, Killick-Kendrick et al. 1989, Endris et al. 1987, Fausto et al. 1992, 1993, Feliciangeli et al. 1993, Rios & Williams, 1995, Ghosh & Mukhopadhway 1996). Up to now, the larval spiracles of sandflies have summarily been described for a limited number of Phlebotomus species (Abonnenc 1972, Maroli et al. 1992). Moreover, no data are available on their ultrastructure and taxonomic significance. In the present investigation, the morphology of the larval spiracles in eight neotropical species [Lutzomyia longipalpis (Lutz & Neiva 1912), L. ovallesi (Ortiz 1952), L. youngi Feliciangeli & Murillo 1987, L. evansi (Nunez-Tovar 1924), L. trinidadensis (Newstead 1922), L. migonei (Franca 1920), L. absonodonta Feliciangeli 1995, and L. venezuelensis (Floch & Abonnenc 1948)] were examined by light and scanning electron microscopy (SEM). Particular attention was given to a comparison among them and with those of other dipterous larvae in order to point out taxonomic and phylogenetic significance in the subfamily Phlebotominae. MATERIALS AND METHODS Fourth instar larvae of L. longipalpis, L. ovallesi, L. youngi, L. evansi , L. trinidadensis, L. migonei, L. absonodonta, and L. venezuelensis used in the present study were obtained from eggs laid by wild females collected in different habitats in Venezuela and reared at the University of Carabobo, Maracay, following the methods described by Killick-Kendrick et al. (1973). For light microscopy, adbominal spiracles of larvae were dissected, mounted on slides in mounting medium and directly observed under Axiophot Zeiss light microscope. Portions of larval abdomens were fixed in Bouin's fixative and embedded in paraffin for histological examination. Sections of 7 µm thickness were stained with toluidine bleu and observed under Axiophot Zeiss light microscope. For SEM, an average of 7 larvae of each species (see Table) were treated with trypsin 0.25% for 5 min and fixed for 2 hr in 4% glutaraldehyde and 5% paraformaldehyde in 0.1 M cacodylate buffer at pH 7.2 (Karnovsky 1965). They were then rinsed overnight in cacodylate buffer, dehydrated in a graded ethanol series, dried by the critical point method using liquid CO2 in a Balzers CPD 020 apparatus, attached to specimen holders, coated with gold in a Balzers Union MED 010 evaporator and observed in a JEOL JMS 5200 electron microscope. RESULTS The fourth instar larva of a sandfly species is amphipneustic, having two pairs of spiracles: the metathoracic pair is situated at the anterior edge of the second thoracic segment and the abdominal one in the posterior corner of the eight abdominal segment (Fig. 1). Although spiracle is smaller, both the thoracic and abdominal spiracles have the same morphological basal plan (Figs 2-5).
Fig. 1: the entire larva shows thoracic (t) and abdominal (a) spiracles. Fig. 2: the thoracic and abdominal spiracles (Fig. 4) are placed at the top of globular bulge (gb). Their structure consists of central (c) and peripheral (p) portions. Fig. 3: a particular of the thoracic and abdominal (Fig. 5) spiracles shows the central portion with chitnous plaques (pl), and the peripheral portion consisting of circularly arranged papillae, separated one another by elongated septa (s). Note the clefts (cl) dividing each papilla in two parts. Fig. 1, bar = 250 um; Figs 2, 4, bars = 3 um; Figs 3, 5, bars = 1um.
In all species studied both thoracic and abdominal spiracles are placed at the top of a globular bulge. Their structure consists of a spiracular plate with a sclerotized central portion and a peripheral portion. The central portion shows chitinous plaques, which vary in number and morphology. The peripheral portion consists of circularly arranged papillae, separated one another by elongated septa (Figs 3, 5). Each papilla is longitudinally crossed by a fine cleft which divides the papilla in two identical parts (Figs 3, 5). The thoracic spiracles always have fewer papillae than the posterior ones. The number of papillae of both thoracic and abdominal spiracles differs from species to species and often between the individuals of the same species. Among the eight species examined, the number of the papillae of the thoracic spiracles varies from six in L. youngi and L. trinidadensis (Figs. 2-5, 6) to nine in L. longipalpis ( Fig. 8). Abdominal spiracles have a minimum of 11 papillae in L. youngi (Fig. 4) and a maximum of 19 in L. longipalpis (Fig. 9). Table shows the number of spiracle papillae (thoracic and abdominal) for each species. Among the species studied, L. longipalpis has the largest thoracic and abdominal spiracular structures (Figs 8, 9), and L. youngi the smallest (Figs 2-5).
The peripheral portion of both thoracic and abdominal spiracles shows two
different features: (i) it appears well defined at the base with papillae
sharply distinct one from the other in L. youngi (Figs 2-4), L.
trinidadensis (Figs 6, 7), L. evansi (
Figures 18-23: Scanning electron micrograph view of
thoracic and abdominal spiracles of Lutzomyia migonei (Figs 18, 19),
bars = 3 um. Light microscopy image of abdominal spiracle in L.
venezuelensis (Fig. 20); it is evident the dark central plug (asterisk)
and the beginning of the tracheal system (arrows), bar = 10 um.
Longitudinal cross sections at different levels of abdominal spiracle of
L. venezuelensis (Figs 21-23); the internal spiracular chamber (ch)
appears to be filled with numerous chitinous projections (arrowheads)
originating from its wall and directed towards the plug, bars = 10mm.
Some variations are also evident in the morphology of the central portion
of the spiracular plates of the various species. In L. youngi ( Figs
2-5) and L. ovallesi (Figs 14, 15) this structure consists of
four similar triangular plaques, with the bases lining the papillae of the
peripheral portion and the apices in the centre of the structure. The
surface of the triangular plaques are more or less convex, so that
depressions are visible among them as well as in the central point where
the plaques converge. In L. trinidadensis (Figs 6, 7), L.
evansi (Figs 10, 11), L. absonodonta (Figs 12, 13) and L.
venezuelensis (Figs 16, 17) the plaques are irregular in shape and not
well lined, so that it is difficult to determine their number. They
converge towards a point that is in an eccentrical position. In L.
migonei, the chitinous central portion of the thoracic spiracle
consists of only one large triangular plaque (Figs 18, 19). In L.
longipalpis, the plaques are not-well evidenced and the central portion
of the spiracular plate looks like a unique chitinous surface with
irregular morphology (Figs 8, 9).
Light microscopy observations of an abdominal spiracle of L.
venezuelensis show a dark central plug which can be seen through the
transparent external chitinous sheet (Fig. 20). The space encircling the
plug connects the peripheral region of the external spiracular plate with
the tracheal system. In longitudinal cross-sections at different levels of
the abdominal spiracle, the central plug consists of electrondense plaques
(Figs 21-23). The spiracular plate is covered by a thin chitinous sheet
that is continuous with the internal spiracular chamber. The latter, that
can be considered a felt chamber, appears to be filled with numerous
chitinous projections originating from its wall and directed towards the
plug (Figs 21-23).
DISCUSSION
As reported by previous authors (Keilin 1944, Whitten 1955, Abonnenc 1972),
the larvae of all Psychodidae are amphipneustic, having a pair of thoracic
and abdominal spiracles. It is now generally accepted that the polypneustic
system, with 8-10 pairs of functional spiracles, characterizes the
terrestrial mode of life.
The oligopneustic (which comprises the amphipneustic type) and apneustic
larvae, with 0-4 pairs of functional spiracles, may be a subsequent
adaptation to a subemerged life in a fluid or semifluid medium. This
condition, derived from the primitive terrestrial polypneustic form, is
considered more specialized (Keilin 1944).
The lack of an intermediate system between the poly and oligopneustic
systems is due to the fact that the first seven pairs of abdominal
spiracles are either all present or all absent. The simultaneous closure of
eight pairs of spiracles (one thoracic and seven abdominal) is probably due
to the partial immersion of the larva in a liquid medium, a factor that
acts more or less uniformly on all segments of the body except the two
terminal ones which are in a position to bring their spiracles in contact
with the air. In different species, these spiracles are modified for
adaptation to a wide diversity of larval habits.
Among Psychodidae, the aquatic larvae of Psychodinae have the
post-abdominal spiracles opening at the end of a respiratory siphon, as an
adaptation to the aquatic mode of life, while the present investigation
shows that larvae of Phlebotominae which live in a decomposed organic
matter are completely devoid of such a syphon. In sandflies, the spiracles
emerge on the top of globular structures and project from the surface of
the larval body for their localization. This localization is probably an
adaptation to life in the decomposed organic matter, which favours the
contact between the spiracles and the air.
In other dipteran species living in similar habitats, for example in the
stratiomyid Metopina rubiceps and Microchrysa polita, the
postabdominal spiracles are found within a pneumatic sac which opens up to
the exterior by means of a transverse slit (Keilin 1944). The larvae having
a pneumatic sac are able to survive by having only intermittent contact
with the air whose admission into the sac is controlled by dilatatory
muscles.
We have no evidence on the mechanism controlling air admission in sandfly
larval spiracles. Light observations of the internal structural arrangement
do not show the presence of muscles or other structures involved in
regulatory function. Further studies need to understand the fine
organization of the spiracular apparatus and clarify the modality of its
regulation.
An evident aspect of the sandfly larval spiracular system is the different
size between the thoracic and abdominal spiracles. In the dipteran larval
spiracular system, postabdominal spiracles are generally the most
developed. In some cases, the thoracic spiracles differ from the
postabdominal not only in structure but also in function. In the dipteran
Gasterophilus larvae, the thoracic spiracles have an internal
occlusion that prevents the entry of extraneous material and help the larva
to survive in the gastro-intestinal tract (Principato & Tosti 1988). Thus
Gasterophilus larvae, appearing to be morphologically amphipneustic,
are functionally metapneustic, having thoracic spiracles with an opening
similar to those of the abdominal spiracles.
Among Psychodinae, some forms have been identified as having a metapneustic
respiratory system. Our observations show that the posterior spiracles of
fourth stage sandfly larvae appear to be almost functioning. They are
characterized by a large internal spiracular chamber that the air probably
reaches by passing through the longitudinal clefts of the peripheral
spiracular plate. The sclerotized plug might be formed by the contraction
and hardening of the chitin surrounding the ecdysial opening through which
the tracheo-spiracular system of the previous larval stage is expelled. The
opening thus becomes obliterated. This spiracle is considered Type II. In
fact, the spiracles in dipterous larvae can be separated into three main
types on the basis of the process of moulting between two successive larval
stages (Keilin 1944). Type I is characterized by spiracles in which the
ecdysial opening becomes the spiracular opening of successive stages. In
Type II, the ecdysial opening is obliterated and a spiracular plate with
clefts papillae are formed around the ecdysial scar which is more or less
central. Type III is similar to Type II except that the spiracular plate
lies outside the ecdysial scar and does not surround it. However, further
studies on the development of the spiracular system throughout the
different sandfly larval stages need to confirm this suggestion.
It is possible that in the sandfly species studied the formation of the
central chitinous scar plug is the result of various processes which
determine four similar triangular plaques in L. youngi, L. absonodonta
and L. ovallesi, some irregular plaques in L. evansi,
L. trinidadensis and L. venezuelensis, only one plaque in L.
migonei and L. longipalpis. In the different species, the
morphology of the spiracles also varies in the number of papillae and in
their aspect as shown in Table. Because of the small
number of species studied, it is not possible at present, to establish any
relationship between the features of the fourth instar larvae spiracles of
phlebotomine sandfly species and their taxonomic status. From these first
observations it seems that the appearance of the peripheral portion of the
spiracles might be a constant character among species of the same subgenus
or species-group. Additionally, the combined number of papillae of the
thoracic and abdominal spiracles might help in distinguishing species of
the same group, which also show a similar structure of the central portion
of the spiracles (e.g. L. ovallesi from L. youngi) (Table).
However, more information is needed on a
greater number of species of the same taxa to reach such a conclusion. On
the other hand, the adaptative significance of the morphological variation
in the spiracular system of different species of phlebotomine sandfly four
instar larvae is, at the moment, hard to understand due to the lack of
information about their microhabitats.
This work was supported by the Commission of the European Community,
Science and Technology for Developing Countries Program
(STD3-TS3*-CT93-0247).
REFERENCES
Copyright 1998 Fundacao Oswaldo Cruz - Fiocruz
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