Mosquito and Sand Fly Gregarines of the Genus

MEEGID 1944 No. of Pages 12, Model 5G 8 May 2014

Infection, Genetics and Evolution xxx (2014) xxx–xxx 1 Contents lists available at ScienceDirect

Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

6 7

3 Mosquito and sand ﬂy gregarines of the genus Ascogregarina and

4 Psychodiella (Apicomplexa: Eugregarinorida, Aseptatorina) – Overview

5 of their taxonomy, life cycle, host speciﬁcity and pathogenicity

a,⇑ b 8 Q1 Lucie Lantova , Petr Volf

9 a Institute of Histology and Embryology, 1st Faculty of Medicine, Charles University in Prague, Albertov 4, 128 00 Prague 2, Czech Republic 10 b Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 44 Prague 2, Czech Republic

11 12 article info abstract 2714 15 Article history: Mosquitoes and sand flies are important blood-sucking vectors of human diseases such as malaria or 28 16 Received 30 January 2014 leishmaniasis. Nevertheless, these insects also carry their own parasites, such as gregarines; these mon- 29 17 Received in revised form 16 April 2014 oxenous pathogens are found exclusively in invertebrates, and some of them have been considered useful 30 18 Accepted 24 April 2014 in biological control. Mosquito and sand fly gregarines originally belonging to a single genus Ascogrega- 31 19 Available online xxxx rina were recently divided into two genera, Ascogregarina comprising parasites of mosquitoes, bat flies, 32 hump-backed flies and fleas and Psychodiella parasitizing sand flies. Currently, nine mosquito Ascogrega- 33 20 Keywords: rina and five Psychodiella species are described. These gregarines go through an extraordinarily interest- 34 21 Ascogregarina ing life cycle; the mosquito and sand fly larvae become infected by oocysts, the development continues 35 22 Psychodiella 23 Coevolution transtadially through the larval and pupal stages to adults and is followed by transmission to the off- 36 24 Host specificity spring by genus specific mechanisms. In adult mosquitoes, ascogregarines develop in the Malpighian 37 25 Pathogenicity tubules, and oocysts are defecated, while in the sand flies, the gregarines are located in the body cavity, 38 26 their oocysts are injected into the accessory glands of females and released during oviposition. These life 39 history differences are strongly supported by phylogenetical study of SSU rDNA proving disparate posi- 40 tion of Ascogregarina and Psychodiella gregarines. This work reviews the current knowledge about Asco- 41 gregarina and Psychodiella gregarines parasitizing mosquitoes and sand flies, respectively. It gives a 42 comprehensive insight into their taxonomy, life cycle, host specificity and pathogenicity, showing a very 43 close relationship of gregarines with their hosts, which suggests a long and strong parasite-host 44 coevolution. 45 Ó 2014 Published by Elsevier B.V. 46 47

48 49

50 1. Introduction et al., 2004), microsporidia (reviewed by Andreadis, 2007), bacteria 63 (reviewed by Minard et al., 2013) and viruses (reviewed by Becnel 64 51 Mosquitoes (Diptera: Culicidae) and sand flies (Diptera: and White, 2007). Similarly, sand flies suffer from the presence of a 65 52 Psychodidae) are blood-sucking insects and important vectors of wide range of pathogens (reviewed by Warburg et al., 1991) such 66 53 human pathogens. Apart from a number of pathogenic viruses, as mites (e.g., Lewis and Macfarlane, 1981), nematodes (e.g., 67 54 mosquitoes transmit Plasmodium (Apicomplexa: Haemosporida) Secundio et al., 2002), protists (e.g., McConnell and Correa, 1964), 68 55 and Filarioidea (Nematoda: Spirurida), while sand flies are the vec- fungi (e.g., Akhoundi et al., 2012), microsporidia (e.g., Matos 69 56 tors of Leishmania (Euglenozoa: Trypanosomatida) and Bartonella et al., 2006), bacteria (e.g., Gouveia et al., 2008) and viruses (e.g., 70 57 (Proteobacteria). As pointed out by Warburg (1991), the life cycle Warburg and Pimenta, 1995). 71 58 of these insects comprising water and terrestrial larval stages, Mosquitoes and sand flies also serve as hosts of gregarines 72 59 respectively, can facilitate growth and persistence of various (Apicomplexa) of the genus Ascogregarina (Ward, Levine and Craig, 73 60 entomopathogens. In mosquitoes, these include mites (e.g., 1982) and Psychodiella Votypka, Lantova and Volf, 2009, respec- 74 61 Kirkhoff et al., 2013), nematodes (reviewed by Petersen, 1980), tively. Phylum Apicomplexa encompasses very important human 75 62 protists (reviewed by Clark, 1980), fungi (reviewed by Scholte pathogens such as Cryptosporidium, Toxoplasma or Plasmodium, 76 while gregarines are parasites exclusively of invertebrates. This 77 might give the impression that they would not be of much 78 ⇑ Corresponding author. Tel.: +420 224968118; fax: +420 224919899. importance to humans; however, especially the neogregarines are 79 E-mail addresses: [email protected] (L. Lantova), [email protected] (P. Volf).

http://dx.doi.org/10.1016/j.meegid.2014.04.021 1567-1348/Ó 2014 Published by Elsevier B.V.

Please cite this article in press as: Lantova, L., Volf, P. Mosquito and sand ﬂy gregarines of the genus Ascogregarina and Psychodiella (Apicomplexa: Eugre- garinorida, Aseptatorina) – Overview of their taxonomy, life cycle, host speciﬁcity and pathogenicity. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/ j.meegid.2014.04.021 MEEGID 1944 No. of Pages 12, Model 5G 8 May 2014

2 L. Lantova, P. Volf / Infection, Genetics and Evolution xxx (2014) xxx–xxx

80 generally considered useful in biological control of insect pests. Fur- included in a single genus Ascogregarina. The type species of this 110 81 thermore, sand fly gregarines are known to seriously harm labora- genus is Ascogregarina culicis (Ross, 1898), originally described as 111 82 tory-reared colonies; therefore, they negatively affect the research Gregarina culicidis (Ross, 1895)orGregarina culicis (Ross, 1898) 112 83 on vector-borne infections. and later renamed as Lankesteria culicis (Wenyon, 1911). The first 113 84 Even though Ascogregarina and Psychodiella are affecting impor- described sand fly gregarine was originally named Monocystis mac- 114 85 tant vectors of human diseases, they have not been given the atten- kiei by Shortt and Swaminath (1927) or Lankesteria phlebotomi 115 86 tion they deserve, and information available about them is mackiei by Missiroli (1932). Grasse (1953) proposed a new name 116 87 currently insufficient. This applies particularly for the sand fly gre- Ascocystis for gregarines of the genus Lankesteria parasitizing 117 88 garines. Before 2010, there had been only three described species insects, and he renamed La. culicis as Ascocystis culicis. Similarly, 118 89 of Psychodiella, the host specificity and pathogenicity of these par- Ormieres (1965) and Tuzet and Rioux (1966) renamed La. ph. mac- 119 90 asites had only been evaluated experimentally in a single study, kiei as Ascocystis mackiei, and later Scorza and Carnevali (1981) 120 91 and closer descriptions of the life cycle and fine structure had been brought morphological evidence placing sand fly gregarines of 121 92 available only about one species. the genus Monocystis into the genus Ascocystis. Levine (1977) and 122 93 This work brings a comprehensive overview of all the described Ormieres (1965) accepted Ascocystis from Grasse (1953) for para- 123 94 species of Ascogregarina and Psychodiella from mosquitoes and sites of Diptera and restricted Lankesteria to parasites of ascidians. 124 95 sand flies, including their taxonomy, life cycle, pathogenicity and However, the name Ascocystis is a synonym for Ascocystis Bather, 125 96 host specificity features. It brings evidence that both species of 1889 used for fossil crinoid echinoderm; therefore, Ward et al. 126 97 these parasites have a very close relationship with their hosts, sug- (1982) established a new name Ascogregarina for the gregarines 127 98 gesting a long coevolution. from Diptera formerly known as Ascocystis. 128 Votypka et al. (2009) pointed out several features that distin- 129 99 2. Taxonomy guish gregarines of the genus Ascogregarina parasitizing mosqui- 130 toes from the ones found in sand flies. Representatives of these 131 100 Members of the genus Ascogregarina (syn. Monocystis von Stein, two groups have different hosts (Culicidae versus Psychodidae), 132 101 1848, Lankesteria Mingazzini, 1891 and Ascocystis Grasse, 1953) and their life cycles differ considerably (for details see Sections 3 133 102 and Psychodiella are aseptate eugregarines (Eugregarinorida: Asep- and 4, Figs. 1 and 2). In adult mosquitoes, ascogregarines develop 134 103 tatorina), and together, they have been recently included in a new in the Malpighian tubules and oocysts are defecated, while in the 135 104 family Ascogregarinidae (Desportes, 2013). The genus Ascogregari- sand flies, the gregarines are located in the body cavity, their 136 105 na comprises species parasitizing mostly Diptera, particularly mos- oocysts are injected into the accessory glands of females and 137 106 quitoes, bat flies (Nycteribiidae) and hump-backed flies (Phoridae) attached to the chorion of oviposited eggs. These life history differ- 138 107 but also fleas (Siphonaptera). The genus Psychodiella is found only ences were strongly supported by phylogenetical study of SSU 139 108 in sand flies. The terminology and history of the final designation of rDNA showing disparate position of mosquito and sand fly grega- 140 109 these parasites is complex. Originally, both these groups were rines; therefore, Votypka et al. (2009) reclassified the genus Asco- 141

Fig. 1. The life cycle of Ascogregarina culicis. (a) Intracellular sporozoite in the larval gut epithelial cell. (b) Young intracellular trophozoites in the larval gut epithelial cells. (c) Gamonts, usually located in the larval gut extracellularly and in the pupal and adult Malpighian tubules. (d) Young gametocyst shortly after formation of syzygy. (e) Mature gametocyst with oocysts. The gametocysts are usually located in the pupal and adult Malpighian tubules. (f) Oocyst with visible sporozoites. The oocysts are usually located in the pupal and adult Malpighian tubules and in faeces.

Fig. 2. The life cycle of Psychodiella chagasi. (a) Extracellular trophozoite attached to the larval gut epithelial cell. (b) Gamonts, usually located in the larval and pupal gut or the pupal and adult body cavity. (c) Syzygy, usually located in the larval gut and adult body cavity. (d) Gametocyst with oocysts, usually located in the larval gut and adult body cavity, in the females attached to the accessory glands. (e) Sporulated oocyst with visible residual body. The oocysts are usually located in the larval gut and faeces, in the adult body cavity, in the lumen of the accessory glands of females and on the exochorion of eggs. Please cite this article in press as: Lantova, L., Volf, P. Mosquito and sand ﬂy gregarines of the genus Ascogregarina and Psychodiella (Apicomplexa: Eugre- garinorida, Aseptatorina) – Overview of their taxonomy, life cycle, host speciﬁcity and pathogenicity. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/ j.meegid.2014.04.021 MEEGID 1944 No. of Pages 12, Model 5G 8 May 2014

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Table 1 Overview of denominated mosquito and sand ﬂy gregarines of the genus Ascogregarina and Psychodiella (originally both included in the genus Ascogregarina).

Name Original name Host species Ascogregarinaa, Ward, Levine and Craig, 1982, mosquito gregarines Ascogregarina culicis (Ross, 1898) type species Gregarina culicis Aedes aegypti (Lin., 1762) As. tripteroidesi (Bhatia, 1938) Lankesteria tripteroidesi Tripteroides doﬂeini (Guenther, 1913) As. barretti (Vavra, 1969) La. barretti Ae. triseriatus (Say, 1823) As. clarki (Sanders and Poinar, 1973) La. clarki Ae. sierrensis (Ludlow, 1905) As. armigerei (Lien and Levine, 1980) Ascocystis armigerei Armigeres subalbatus (Coquillett, 1898) As. lanyuensis (Lien and Levine, 1980) Asc. lanyuensis Ae. alcasidi (Huang, 1972) As. taiwanensis (Lien and Levine, 1980) Asc. taiwanensis Ae. albopictus (Skuse, 1894) As. geniculati (Munstermann and Levine, 1983) As. geniculati Ae. geniculatus (Olivier, 1791) As. polynesiensis (Levine, 1985) As. polynesiensis Ae. polynesiensis (Marks, 1951) Psychodiellaa, Votypka, Lantova and Volf, 2009, sand ﬂy gregarines Psychodiella mackiei (Shortt and Swaminath, 1927) Monocystis mackiei Phlebotomus argentipes (Ann. and Brun., 1908) Ps. chagasi (Adler and Mayrink, 1961) type species M. chagasi Lutzomyia longipalpis (Lutz and Neiva, 1912) Ps. saraviae (Ostrovska, Warburg and Montoya-Lerma, 1990) As. saraviae L. lichyi (Floch and Abonnenc, 1950) Ps. sergenti (Lantova, Volf and Votypka, 2010) Ps. sergenti Ph. sergenti (Parrot, 1917) Ps. tobbi (Lantova, Volf and Votypka, 2010) Ps. tobbi Ph. tobbi (Adler, Theodor and Lourie, 1930)

a Additional possible Ascogregarina and Psychodiella species that have not been denominated or species not parasitizing sand ﬂies and mosquitoes are listed in the text (see Sections 3.11 and 4.7).

142 gregarina, divided it into two and established a new genus accom- 3. Genus Ascogregarina: the life cycle and species overview 187 143 modating sand fly gregarines – Psychodiella. The status of the tax- 144 onomy of mosquito and sand fly gregarines of the former genus 3.1. General life cycle 188 145 Ascogregarina with nine described mosquito gregarine species 146 and five described sand fly gregarine species is presented in The life cycle of As. culicis (Fig. 1), the type species of the genus 189 147 Table 1. Ascogregarina, is very similar to other mosquito ascogregarines and 190 148 Information about molecular taxonomy of sand fly and mosquito is used as a typical example. Minor interspecific differences and 191 149 gregarines is currently lacking. Genes for SSU rRNA were sequenced details are explained for each species separately (see Sections 192 150 for Ascogregarina armigerei (Lien and Levine, 1980), Ascogregarina 3.2–3.11). Published measurements of oocysts of all mosquito 193 151 sp. from Ochlerotatus japonicus japonicus, As. culicis and Ascogregari- Ascogregarina species are summarized in Table 2. 194 152 na taiwanensis (Lien and Levine, 1980)byRoychoudhury et al. The mosquito larvae become infected by ingesting Ascogregarina 195 153 (2007a), and these authors showed close relationship of ascogrega- oocysts. Each spindle-shaped oocyst contains eight sporozoites, 196 154 rines and cryptosporidians. Partial sequences of SSU rDNA and 28S which are released in the larval intestine and invade the epithelial 197 155 rDNA and sequences of ITS1, 5.8S rDNA and ITS2 of Ascogregarina cells. Inside the cells, the sporozoites develop into trophozoites, 198 156 barretti (Vavra, 1969), As. culicis and As. taiwanensis were submitted which are later, when the epithelial cell ruptures, released into 199 157 by Morales et al. (2005). Furthermore, there are directly submitted the gut lumen. Initially, they attach to the epithelial cells of the lar- 200 158 sequences of actin gene, partial SSU rDNA and ITS1, 5.8S rDNA, ITS2, val intestine, and during pupation, the gregarines migrate to the 201 159 26S rDNA and 5S rDNA of As. taiwanensis. Templeton et al. (2010) Malpighian tubules, the site of sexual development of the parasites 202 160 accomplished a whole-genome-sequence survey for As. taiwanensis. in adult mosquitoes; the gamonts pair in syzygies and develop into 203 161 Less information is available about Psychodiella gregarines; there gametocysts with oocysts inside. The oocysts are released during 204 162 are only two studies revealing the sequences of SSU rDNA of Psych- defecation with faeces into the water and infect newly hatched lar- 205 163 odiella sergenti Lantova, Volf and Votypka 2010, Psychodiella chagasi vae. (McCray et al., 1970; Vavra, 1969; Walsh and Callaway, 1969; 206 164 (Adler and Mayrink, 1961) and Psychodiella tobbi Lantova, Volf and Wenyon, 1911). 207 165 Votypka, 2010 (Lantova et al., 2010; Votypka et al., 2009). The species of the genus Ascogregarina are listed below accord- 208 166 The very complex history of the terminology and the final des- ing to the year of their description. 209 167 ignation of the genus Psychodiella and Ascogregarina show that the 168 life cycle and morphological characteristics are not sufficient any- 169 more for determining the genera and species of gregarines. 3.2. As. culicis 210 170 Description of new gregarine species should combine both the bio- 171 logical and molecular features, as shown by Lantova et al. (2010), As. culicis was described in India by Ross (1895). Oocysts of this 211 172 Leander et al. (2003a), Rueckert and Leander (2009) or Votypka parasite are infective to all larval stages of Aedes aegypti (Lin., 212 173 et al. (2009). The lack of taxonomical data about Psychodiella and 1762); when the early larval instars are infected with the gregarine 213 174 Ascogregarina calls for more molecular phylogenetic studies and oocysts, the development of both the parasite and the host are 214 175 sequencing more genes of more species to reveal the hidden biodi- synchronized, while the gregarine development stops at the 215 176 versity among sand fly and mosquito gregarines. Such a complex gamont stage, when the oocysts are ingested by the late 4th instar 216 177 approach would also support the recent inclusion of Ascogregarina larvae. Similar results were observed also for As. taiwanensis in 217 178 and Psychodiella in a new family Ascogregarinidae (Desportes, Aedes albopictus (Skuse, 1894) (Roychoudhury and Kobayashi, 218 179 2013) and would clarify the relationship of siphonapteran and 2006; see Sections 3.8). 219 180 brachyceran Ascogregarina species with nematoceran Ascogregarina The sporozoites are 9.5–10 lm long with a tapered posterior 220 181 and Psychodiella species. Furthermore, the close relationship of end. Their pellicle, according to the authors (Sheffield et al., 221 182 aseptate eugregarines and neogregarines as demonstrated e.g., by 1971), consists of an outer and a thicker inner membrane. The 222 183 Carreno et al. (1999), Lantova et al. (2010), Leander et al. anterior part of the sporozoite contains conoid, two apical rings 223 184 (2003a,b, 2006) and Votypka et al. (2009) may raise the question, and a polar ring. A ‘‘flask-shaped’’ organelle observed in the ante- 224 185 whether the absence of merogony in some aseptate eugregarines rior part of mostly extracellular sporozoites was suggested to have 225 186 is not only the case of the merogony not being detected. a function in the host cell invasion (Sheffield et al., 1971). Tropho- 226 Please cite this article in press as: Lantova, L., Volf, P. Mosquito and sand fly gregarines of the genus Ascogregarina and Psychodiella (Apicomplexa: Eugre- garinorida, Aseptatorina) – Overview of their taxonomy, life cycle, host specificity and pathogenicity. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/ j.meegid.2014.04.021 MEEGID 1944 No. of Pages 12, Model 5G 8 May 2014

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Table 2 Overview of published oocyst measurements of mosquito Ascogregarina and sand ﬂy Psychodiella species.

Gregarine species Oocyst length Oocyst width References Ascogregarina culicis 6 lm Not given Ross (1895) 10 lm6lm Ray (1933) 10–12 lm 6–7 lm Vavra (1969) 11 lm5lm Lien and Levine (1980) 11.2 (9.5–12.2) lm 4.9 (4.7–5.1) lm Dellape et al. (2005) 11.1 (10.6–11.4) lm 5 (4.8–5.5) lm Vezzani and Wisnivesky (2006) 8.8 lm 4.22 lm Roychoudhury et al. (2007a) As. tripteroidesi Not published Not published As. barretti 11 lm 5.4–5.7 lm Vavra (1969) As. clarki 11 (10–12) lm 6 (5–6) lm Sanders and Poinar (1973) As. armigerei 14.5 lm6lm Lien and Levine (1980) 13.2 lm 5.78 lm Roychoudhury et al. (2007a) As. lanyuensis 9 lm5lm Lien and Levine (1980) As. taiwanensis 10 lm5lm Lien and Levine (1980) 9.3 (8.3–9.9) lm 4.6 (4.3–4.9) lm Garcia et al. (1994) 8.72 lm 4.97 lm Chen et al. (1997a) 9.9 lm 4.85 lm Roychoudhury et al. (2007a) As. geniculati 12 lm4lm Ganapati and Tate (1949) 9–11 lm 4–6 lm Kramar (1952) 13 (12–14) lm5lm Munstermann and Levine (1983) As. polynesiensis 9.32 lm 4.24 lm Pillai et al. (1976) Psychodiella mackiei 9.6 lm 5.8 lm Shortt and Swaminath (1927) Ps. chagasi 10.9–11.9 lm 5.8 lm Adler and Mayrink (1961) 10.9 (10–12) lm 6.5 (6–7) lm Brazil and Ryan (1984) 12.7 (11.6–14.5) lm 7.5 (7.2–7.8) lm Ostrovska et al. (1990) 12.7 (11.6–14.5) lm 7.5 (7.2–7.8) lm Warburg and Ostrovska (1991) 12.7 (12–13.3) lm 8.3 (7.3–8.9) lm Lantova et al. (2010) Ps. saraviae 12.4 (11.6–13.1) lm 5.8 (5.6–5.9) lm Ostrovska et al. (1990) 12.4 lm 5.8 lm Warburg (1991) Ps. sergenti 9.6 (8.7–10.3) lm 6.7 (6.2–7.1) lm Lantova et al. (2010) Ps. tobbi 9.6 (8.8–10.7) lm 7.5 (6.8–8.5) lm Lantova et al. (2010)

227 zoites of As. culicis are 170 lm long, gametocysts are 71–125 lmin 3.4. As. barretti 258 228 diameter, and oocysts are 11 lm long and 5 lm wide (Lien and 229 Levine, 1980). As. barretti was described from Aedes triseriatus (Say, 1823) in 259 230 Susceptibility of Ae. aegypti to As. culicis varies among geograph- Texas by Vavra (1969). Trophozoites develop in the epithelial cells 260 231 ical strains (Reyes-Villanueva et al., 2003; Sulaiman, 1992); fur- of the larval intestine, after reaching the size of 150–200 lm, they 261 232 thermore, one strain of Ae. aegypti from Trinidad was not are released from the ruptured cells and appear in the ectoperi- 262 233 susceptible to a Florida strain of As. culicis as no oocysts developed, trophic space of the intestine as gamonts. These grow up to the 263 234 while other Trinidad strains were susceptible (Beier et al., 1995). length of 310 lm, and during pupation, they enter into the Malpi- 264 235 Several studies on the prevalence of As. culicis in natural Ae. ghian tubules. Gametocysts are 60–100 lm in diameter, and 265 236 aegypti populations showed that it ranges from 0% to 100% and is oocysts with eight sporozoites measure 11 Â 5.4–5.7 lm. This 266 237 seasonally and spatially very heterogeneous (Albicocco and gregarine differs from As. culicis in several features: the location 267 238 Vezzani, 2009; Beier et al., 1995; Blackmore et al., 1995; Dellape of the trophozoites within the epithelial cells, the position of their 268 239 et al., 2005; Vezzani and Wisnivesky, 2006). nucleus, the character of longitudinal folds, the presence of a 269 240 According to some authors, As. culicis was found also in mosquito mucron and the size of paraglycogen granules (Vavra, 1969). 270 241 species other than Ae. aegypti: Kramar (1952) and Ganapati and According to various authors (Fukuda et al., 1997; Porter and 271 242 Tate (1949) reported it in Aedes geniculatus (Olivier, 1791), Ray DeFoliart, 1985; Siegel et al., 1992; Van Rhein et al., 2000), the 272 243 (1933) in Ae. albopictus, Feng (1930) in Aedes koreicus and Pillai prevalence of this parasite ranges between 3% and 100%, it is sea- 273 244 et al. (1976) in Aedes polynesiensis Marks, 1951. Vavra (1969) sonally and spatially heterogeneous and dependent on the parity of 274 245 suggested that only those from Ae. albopictus and Ae. koreicus could females. 275 246 be As. culicis. Munstermann and Levine (1983) consider the two 247 gregarines from Ae. geniculatus to be Ascogregarina geniculati 276 248 Munstermann and Levine, 1983, and Levine (1985) designated the 3.5. As. clarki 249 gregarine from Ae. polynesiensis as Ascogregarina polynesiensis 277 250 Levine, 1985 (see Sections 3.9 and 3.10). Ascogregarina clarki (Sanders and Poinar, 1973) was described from Aedes sierrensis (Ludlow, 1905) in California by Sanders and 278 Poinar (1973). Several features distinguish this gregarine from As. 279 251 3.3. As. tripteroidesi culicis and As. barretti: the character of gamonts and their nucleus, 280 the structure and position of residual bodies in the oocysts and the 281 252 As. tripteroidesi (Bhatia, 1938) was found in Tripteroides dofleini fact that the trophozoites are always intracellular in the anterior 282 253 (Guenther, 1913) in Sri Lanka by Guenther (1914) and later part of the larval midgut. Sporozoites measure 8 Â 1–2 lm, mature 283 254 denominated by Bhatia (1938). The whole life cycle was not trophozoites measure 129.1 Â 26 lm, and they have unresolved 284 255 described; the only known stages are trophozoites, which were number of membranes in the pellicle. According to the authors, 285 256 recorded in the body cavity, trachea and anal gills of mosquito the passage of the trophozoites to the Malpighian tubules is rather 286 257 larvae. passive, as the release of the gregarines from the epithelial cells is a 287 Please cite this article in press as: Lantova, L., Volf, P. Mosquito and sand fly gregarines of the genus Ascogregarina and Psychodiella (Apicomplexa: Eugre- garinorida, Aseptatorina) – Overview of their taxonomy, life cycle, host specificity and pathogenicity. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/ j.meegid.2014.04.021 MEEGID 1944 No. of Pages 12, Model 5G 8 May 2014

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288 result of natural histolysis of the midgut cells during pupation. that can differentiate this species from other mosquito ascogrega- 346 289 Gamonts measure 226 Â 31 lm, gametocysts are 78 lm in diame- rines is the dimension of oocysts that measure 13.5 Â 5 lm. 347 290 ter on average, and oocysts are 11 lm long and 6 lm wide Gamonts measure on average 175 Â 31 lm, and gametocysts are 348 291 (Sanders and Poinar, 1973). 77 lm in diameter (Munstermann and Levine, 1983). 349

292 3.6. As. armigerei 3.10. As. polynesiensis 350

293 As. armigerei was described from Armigeres subalbatus (Coquil- As. polynesiensis was found in Ae. polynesiensis in Samoa by Pillai 351 294 lett, 1898) in Taiwan by Lien and Levine (1980). The trophozoites et al. (1976) and was, similarly to As. geniculati, originally described 352 295 measure 135 Â 23 lm and are contractile, the gametocysts are as As. culicis. However, Levine (1985) pointed out that As. culicis dif- 353 296 71.2 lm in diameter. The character and size of the oocysts fers from the gregarine from Ae. polynesiensis, and that Pillai et al. 354 297 (14.5 Â 6 lm) clearly distinguishes between As. culicis, As. armig- (1976) examined 325 Ae. aegypti (the natural host of As. culicis) and 355 298 erei, Ascogregarina lanyuensis (Lien and Levine, 1980) and As. tai- found only a single infected one, while the prevalence in Ae. 356 299 wanensis (Lien and Levine, 1980). polynesiensis was 39.5%. Therefore, this gregarine was renamed to 357 As. polynesiensis (Levine, 1985). Trophozoites have a mean dimen- 358 300 3.7. As. lanyuensis sion of 65 Â 35 lm, gametocysts measure approximately 40 lm, 359 and oocysts measure 9.32 Â 4.24 lm(Pillai et al., 1976). 360 301 As. lanyuensis was described from Aedes alcasidi Huang, 1972 in 302 Taiwan along with two other Ascogregarina species (Lien and 3.11. Other Ascogregarina species 361 303 Levine, 1980). Trophozoites measure 190 Â 26 lm, gametocysts 304 are 89.9 lm in diameter, and oocysts are 9 lm long and 5 lm wide. A gregarine similar to As. barretti was found in Indiana in Aedes 362 hendersoni (Rowton et al., 1987); however, the differences in the 363 305 3.8. As. taiwanensis number of gregarine stages, localization of the trophozoites within 364 the larval gut and the presence of dead gamonts in cross-infections 365 306 As. taiwanensis was described from Ae. albopictus in Taiwan by with As. barretti and Ae. triseriatus suggest that the gregarine from 366 307 Lien and Levine (1980). Sporozoites are slender with a three-lay- Ae. hendersoni is a new species, distinct from As. barretti. This was 367 308 ered pellicle (recorded also in trophozoites) and possess typical accepted by Chen (1999). 368 309 apical complex with conoid, polar rings, rhoptries, subpellicular A gregarine described from O. j. japonicus in Japan by 369 310 microtubules and micronemes (Chen et al., 1997a). The amylopec- Roychoudhury et al. (2007b) was originally named as ‘‘Ascogregarina 370 311 tin granules recorded in the oocysts disappear during the morpho- japonicus’’; however, the same authors denominated it as 371 312 genesis of the sporozoites (Chen et al., 1997a). Chen et al. (1997b) Ascogregarina sp. from O. j. japonicus (Roychoudhury et al., 2007a). 372 313 usually found two sporozoites in each epithelial cell. The release of The only described stage of this species are the oocysts, which mea- 373 314 the sporozoites from the oocysts may be triggered by V-ATPase sure on average 10.7 Â 5.15 lm(Roychoudhury et al., 2007a). 374 315 modulated alkalization in the anterior midgut of the mosquito, Other species of the genus Ascogregarina have been recorded 375 316 and V-ATPase may also play a role in the parasite invasion and from different mosquito species in the USA, Brazil, West Africa, 376 317 the formation of the extracellular stages (Huang et al., 2006). China, Malaysia, Philippines, Italy or France (reviewed by 377 318 The development of As. taiwanensis is influenced by the larval Christophers, 1960; Clark, 1980). 378 319 age at the time of infection in a similar pattern as in As. culicis Additionally to those parasitizing mosquitoes, there are three 379 320 (Roychoudhury and Kobayashi, 2006; see Sections 3.2). The devel- Ascogregarina species parasitizing hosts belonging to other orders 380 321 opment of the extracellular trophozoites is conditioned by their or suborders. Ascogregarina cheopisi Mourya, Geevarghese and 381 322 migration to the Malpighian tubules; the ones who fail to migrate Gokhale, 1996 was described from the flea (Siphonaptera: 382 323 undergo apoptosis (Chen et al., 2013). At the anterior end of the Pulicidae) Xenopsylla cheopis (Rothschild, 1903) in India by 383 324 migrating parasite, a ‘‘protruding apparatus’’ with enhanced actin Mourya et al. (1996). Ascogregarina galliardi (Garnham, 1973) was 384 325 expression is formed (Chen and Fan-Chiang, 2001). The sexual described from the bat fly (Brachycera: Nycteribiidae) Nycteribia 385 326 reproduction of As. taiwanensis in the Malpighian tubules is syn- dentata Theodor, 1967 by Garnham (1973), and Purrini (1980) 386 327 chronized with the host metamorphosis; increase of the level of identified and described Ascogregarina brachyceri (Purrini, 1980) 387 328 the molting hormone 20-hydroxyecdysone signals migration of in the hump-backed fly (Brachycera: Phoridae) Megaselia subnitida 388 329 the parasite and expedites the formation of gametocysts (Chen, Lundbeck, 1920 in India. 389 330 1999; Chen and Yang, 1996). Gamonts are 234 lm long, gamet- 331 ocysts are 87.5 lm in diameter, and oocysts measure 10 Â 5 lm 332 (Lien and Levine, 1980). 4. Genus Psychodiella: the life cycle and species overview 390 333 The prevalence of this parasite was recorded to be up to 100% 334 by various authors (Blackmore et al., 1995; Dos Passos and Tadei, 4.1. General life cycle 391 335 2008; Garcia et al., 1994; Munstermann and Wesson, 1990; 336 Reyes-Villanueva et al., 2013). It was found to be the most common The life cycle of Ps. chagasi (Fig. 2), the type species of the genus 392 337 parasite of Ae. albopictus in Florida (Fukuda et al., 1997), and in Psychodiella, is used as a typical example. Details and interspecific 393 338 New Orleans, the prevalence was shown to be seasonally heteroge- differences are explained for each species separately (see Sections 394 339 neous (Comiskey et al., 1999a). 4.2–4.7). Published measurements of oocysts of all Psychodiella 395 species are summarized in Table 2. 396 340 3.9. As. geniculati The first instar larvae are infected by swallowing spindle- 397 shaped oocysts. Eight sporozoites released from these oocysts 398 341 As. geniculati was found in Ae. geniculatus by Ganapati and Tate reside in the larval midgut, attach to the epithelial cells and 399 342 (1949) in England and by Kramar (1952) in the former develop into trophozoites. Later, gamonts can be found mostly in 400 343 Czechoslovakia. It was originally described as As. culicis; however, the larval gut lumen, where the gregarines undergo sexual devel- 401 344 Munstermann and Levine (1983), who were studying this gregarine opment from the formation of syzygies to the production of 402 345 in Sardinia, determined it as As. geniculati. The main characteristic oocysts. In the adults, the gregarines are located in the body cavity 403 Please cite this article in press as: Lantova, L., Volf, P. Mosquito and sand fly gregarines of the genus Ascogregarina and Psychodiella (Apicomplexa: Eugre- garinorida, Aseptatorina) – Overview of their taxonomy, life cycle, host specificity and pathogenicity. 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404 forming syzygies and gametocysts with oocysts inside. The gamet- cycle features that differentiate this parasite from Ps. mackiei.In 466 405 ocysts attach to the accessory glands of females, and the oocysts the 1st instar larvae, the sporozoites and young trophozoites are 467 406 are injected into their lumen. This unique mechanism of vertical attached to the larval epithelium through an osmiophilic contact 468 407 transmission was recorded by several authors (Adler and zone; they are never located intracellularly (Warburg and 469 408 Mayrink, 1961; Coelho and Falcao, 1964; Lantova and Volf, 2012; Ostrovska, 1991). The sporozoites are surrounded by a two-layered 470 409 Lewis et al., 1970; Scorza and Carnevali, 1981) and supports the pellicle with subpellicular microtubules and possess an apical 471 410 hypothesis about coevolution of gregarines and sand flies complex with conoid. The oval gamonts (60–90 lm), on the other 472 411 (Ostrovska et al., 1990). During oviposition, contents of the glands hand, have a three-layered pellicle forming longitudinal epicytic 473 412 including the oocysts are attached to the chorion of eggs and serve folds (Warburg and Ostrovska, 1991). They were found in the ecto- 474 413 as a source of infection for newly hatched larvae (Adler and peritrophic space of the intestine of the 3rd and 4th instar larvae 475 414 Mayrink, 1961; Coelho and Falcao, 1964; Warburg and Ostrovska, by Warburg and Ostrovska (1991) and in the ectoperitrophic space 476 415 1991). This general life cycle is modified in Psychodiella mackiei of the 1st, 2nd and 3rd and in the intestinal lumen of the 4th instar 477 416 (Shortt and Swaminath, 1927), where the development of the spor- larvae (Coelho and Falcao, 1964). The gregarine is able to complete 478 417 ozoites and trophozoites in larvae is intracellular (Shortt and its life cycle in the larvae, and oocysts in their faeces are a source of 479 418 Swaminath, 1927; see Section 4.2). horizontal transmission (Coelho and Falcao, 1964). 480 419 The species of the genus Psychodiella are listed below according In pupae, only gamonts were found (Coelho and Falcao, 1964), 481 420 to the year of description. and the relocation from the midgut to the body cavity occurs dur- 482 ing pupation (Shortt and Swaminath, 1927; Warburg and 483 421 4.2. Ps. mackiei Ostrovska, 1991). In males, Adler and Mayrink (1961) recorded 484 only gamonts, while Coelho and Falcao (1964) found all the devel- 485 422 Ps. mackiei was described by Shortt and Swaminath (1927) from opmental stages of Ps. chagasi (gamonts, syzygies, gametocysts and 486 423 Phlebotomus argentipes Ann. and Brun., 1908 in India and later by oocysts) in the body cavity and a few gamonts also in the intestine. 487 424 Missiroli (1929, 1932) from Phlebotomus papatasi in Italy. It is the In females, Adler and Mayrink (1961) found syzygies, gamet- 488 425 only Psychodiella species with known intracellular development. ocysts and oocysts of Ps. chagasi 2–4 days post blood meal, while 489 426 In the larvae, four gregarine stages can be found: ingested oocysts, Warburg and Ostrovska (1991) found them 72 h post blood meal. 490 427 sporozoites released from these oocysts, intracellular stages (all in The smallest gamonts are 30 lm in diameter (those over 60 lm have 491 428 the intestine) and adult gregarines in the intestine (often in the a proboscis-like projection), and adult round or oval gamonts which 492 429 ectoperitrophic space) or the body cavity. The sporozoites (leaf- are ready to undergo syzygy measure 72–120 lm in diameter. Their 493 430 shaped, 4.8 lm long and 1.8 lm wide) are located mostly in the nucleus (28 lm) has a distinctive nucleolus (8–10 lm) (Adler and 494 431 posterior part of the midgut. The authors postulate that most spor- Mayrink, 1961). According to various authors, gametocysts range 495 432 ozoites do not survive defecation; therefore, only a certain number from 72 to 150 lm in diameter (Adler and Mayrink, 1961; Lantova 496 433 of them are able to invade the host epithelial cells. The intracellular et al., 2010; Warburg and Ostrovska, 1991). According to Adler and 497 434 gregarines (23.4 lm) are initially round and later become triangular Mayrink (1961), oocysts measure 10.9–11.9 Â 5.8 lm. The cyto- 498 435 with no special organ of attachment. They concentrate mostly in the plasm of the developing oocyst contains amylopectin granules, 499 436 posterior intestine, and they are released from the epithelial cells and all the life cycle stages apart from the gametocysts have a diffuse 500 437 into either the intestinal lumen or the body cavity. The gamonts actin-like protein in the cytoplasm suggesting the involvement of 501 438 (round, oval or pear-shaped, measuring 101.4 Â 78 lm) with a dis- actin in the movement (Warburg and Ostrovska, 1991). 502 439 tinct nucleus (30 lm) and nucleolus (8–10 lm) are motile with well The gametocysts are attached to the accessory glands of 503 440 marked longitudinal striations (Shortt and Swaminath, 1927). females. Injection of the oocysts into the accessory glands is 504 441 In the pupae, only gamonts of Ps. mackiei occur, mostly in the enhanced by plasmatocyte humoral encapsulation; the encapsu- 505 442 posterior body cavity (Shortt and Swaminath, 1927). In the adults, lated gametocysts and the internal pressure of the developing 506 443 all the parasite life stages including oocysts were found. The large oocysts results in the rupture of the gametocysts at the site where 507 444 fully grown gamonts are located always in the body cavity, often in they are attached to the accessory glands, releasing the oocysts 508 445 the posterior part. However, they are never within the digestive into the gland lumen (Warburg and Ostrovska, 1989). The majority 509 446 tract, and they tend to cluster around the reproductive organs. Sex- of the oocysts in the accessory glands are uninucleate, and the 510 447 ual development of the gregarines occurs irrespective of a blood nuclear divisions occur after 24 h in a moist chamber (Adler and 511 448 meal intake. Gametocysts are 66.3–152.1 lm in diameter, spheri- Mayrink, 1961). There are two modes of vertical transmission of 512 449 cal or oval, and they are mostly attached to the common oviduct. this gregarine; the larvae ingest either the oocysts released from 513 450 Oocysts are broad spindle-shaped, 9.6 lm long and 5.8 lm wide, the accessory glands on the exochorion of eggs or the ones in the 514 451 with knob-like projection at each pole. The pressure produced by body cavity of dead sand flies. 515 452 the growth of oocysts and the gametocysts causes disruption of The prevalence of Ps. chagasi in wild-caught L. longipalpis in 516 453 the gametocyst wall at the site where they are attached to the ovi- Brazil was recorded by Adler and Mayrink (1961) as 10%, while 517 454 duct, and the oocysts are injected into its lumen (Shortt and the infection rate of laboratory-reared colonies varies between 518 455 Swaminath, 1927). Wu remarks (as unpublished observation in 86% and 100% (Dougherty and Ward, 1991; Warburg and 519 456 Ostrovska et al., 1990) that Ps. mackiei oocysts were also found in Ostrovska, 1989; Wu and Tesh, 1989). 520 457 the accessory glands. In Brazil, gregarines identified as Ps. chagasi were recorded from 521 458 The prevalence of infected sand flies was roughly 25% or less in several sand fly species: Lutzomyia townsendi (Scorza and Carnevali, 522 459 nature, while in the laboratory colonies, where the chance of infec- 1981), Lutzomyia evandroi (Brazil and Ryan, 1984), Lutzomyia sallesi 523 460 tion is much higher, it was almost 100% (Shortt and Swaminath, (Coelho and Falcao, 1964), Lutzomyia sordelli (Oliveira et al., 1991 in 524 461 1927). Brazil et al., 2002) and Lutzomyia cruzi (Brazil et al., 2002). 525

462 4.3. Ps. chagasi 4.4. Psychodiella saraviae 526

463 Ps. chagasi, originally named M. chagasi, was described from Ps. saraviae (Ostrovska, Warburg and Montoya-Lerma, 1990) was 527 464 Lutzomyia longipalpis (Lutz and Neiva, 1912) females in Brazil by described from females of Lutzomyia lichyi (Floch and Abonnenc, 528 465 Adler and Mayrink (1961), who gave several morphological and life 1950) in Colombia (Ostrovska et al., 1990). Two or three gametocysts 529 Please cite this article in press as: Lantova, L., Volf, P. Mosquito and sand ﬂy gregarines of the genus Ascogregarina and Psychodiella (Apicomplexa: Eugre- garinorida, Aseptatorina) – Overview of their taxonomy, life cycle, host speciﬁcity and pathogenicity. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/ j.meegid.2014.04.021 MEEGID 1944 No. of Pages 12, Model 5G 8 May 2014

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530 were attached to the accessory glands of each infected female. (Lantova et al., 2010). In the 4th instar larvae, the gamonts and 592 531 Oocysts (12.4 Â 5.8 lm) are located in the gametocysts, in the glands gametocysts are mostly located in the ectoperitrophic space (in 593 532 or on the egg surface. The differences in the size and shape of the younger individuals) or the body cavity (in older individuals, 594 533 oocysts (those of Ps. saraviae have thinner walls and narrower mid- shortly before pupation). In the adults, the gamonts and gamet- 595 534 sections than those of Ps. chagasi) give evidence that Ps. saraviae is a ocysts occur in the body cavity, and the oocysts are located also 596 535 different species (Ostrovska et al., 1990). The prevalence of this par- in the accessory glands. The round or oval gamonts measure on 597 536 asite in Colombia was 1.9–3.3% (Ostrovska et al., 1990). average 123.6 lm, gametocysts measure 137.2 lm, and broad 598 537 Oliveira et al. (1991) in Brazil et al. (2002) found a gregarine in spindle-shaped oocysts measure 9.6 Â 7.5 lm. The prevalence in 599 538 Lutzomyia schreiberi, which they consider to be Ps. saraviae. the wild-caught sand ﬂies was 16.8% (Lantova et al., 2010). 600

539 4.5. Ps. sergenti 4.7. Other Psychodiella species 601

540 Ps. sergenti was described from Phlebotomus sergenti Parrot, A gregarine found in L. v. occidentis in California (Ayala, 1971) 602 541 1917 laboratory-reared colony originating from Turkey (Lantova differs from Ps. chagasi in several morphological and life cycle char- 603 542 et al., 2010). This gregarine was distinguished from Ps. chagasi acteristics suggesting that it is a new species; gamonts are pear- 604 543 and Ps. tobbi (see Section 4.6) due to the differences in their life shaped (74–180 Â 58–95 lm) or spherical (68–180 lm), with a 605 544 cycles and the size and morphology of their life stages, due to their light nucleus (24–26 lm) and nucleolus (8.5 lm), gametocysts 606 545 high host specificity in cross-infections (see Section 5) and due to are spherical (110–190 lm), and oocysts are spindle-shaped 607 546 the differences in the sequences of SSU rDNA. The prevalence of (10.5 Â 6 lm) (Ayala, 1971). 608 547 Ps. sergenti in wild-caught sand flies was 15.6% (Lantova et al., A number of studies recorded unidentified gregarine species 609 548 2010), while in the colony, it reached 100% (Lantova, unpublished). from Lutzomyia shannoni in Belize (Garnham and Lewis, 1959), 610 549 The life cycle of Ps. sergenti possesses a unique feature, similar Lutzomyia cruciata in Belize (Lewis, 1965), Lutzomyia flaviscutellata 611 550 to the one recorded in a single study of an undescribed gregarine in Brazil (Lewis et al., 1970), six species of Lutzomyia sp. in Brazil 612 551 parasite in Lutzomyia vexatrix occidentis by Ayala (1971) (see (Mayrink et al., 1979), ten species of Phlebotomus sp. in Panama 613 552 Section 4.7). In adult Ph. sergenti, the sexual development (forma- (McConnell and Correa, 1964) and Lutzomyia apache in Wyoming 614 553 tion of syzygies, gametocysts and oocysts) occurs exclusively in (Reeves et al., 2008). 615 554 blood-fed females, while in males and unfed females, only gamonts 555 were recorded (Lantova and Volf, 2012; Lantova et al., 2010). 556 Contrastingly, e.g., Ps. chagasi (Coelho and Falcao, 1964)orPs. tobbi 5. Host specificity 616 557 (Lantova et al., 2010; see Section 4.6) develop into syzygies, gamet- 558 ocysts and oocysts also in males and unfed females. Ayala (1973) The most studied mosquito gregarine in terms of its host spec- 617 559 and Lantova and Volf (2012) hypothesize that the hormonal ificity is As. taiwanensis. This parasite infected 100% of Ae. aegypti 618 560 changes influenced by a blood meal intake trigger the sexual cycle and Aedes taeniorhynchus larvae (by oocysts recovered from Ae. 619 561 of the gregarines in Ph. sergenti and L. v. occidentis, respectively. taeniorhynchus adults); however, it was not infective to Culex and 620 562 Lantova et al. (2011) determined the number of oocysts com- Anopheles species (Garcia et al., 1994). Munstermann and Wesson 621 563 monly produced in a laboratory-reared colony of Ph. sergenti as (1990) found As. taiwanensis in Aedes epactius and Culex restuans, 622 564 15,158 oocysts per one Ph. sergenti female. The oocysts are located and after experimental infections, oocysts were recovered from 623 565 on the chorion of eggs, in contact with the exochorion longitudinal Ae. aegypti and Aedes atropalpus. Lien and Levine (1980) were also 624 566 sculpturing ridges, suggesting that the process of the attachment of able to recover As. taiwanensis oocysts from its unnatural host, 625 567 the oocysts is connected to the formation of exochorion (Lantova experimentally infected Ae. alcasidi. Furthermore, this species 626 568 and Volf, 2012). Sporozoites, located in the ectoperitrophic space infected sabethine mosquito Wyeomyia smithii, and although the 627 569 of the 1st instar larval midgut, are never intracellular and have a infection rates were low, gametocysts were recovered from one 628 570 three-layered pellicle, distinctive mucron, long conoid, numerous female (Reeves and McCullough, 2002). A recent study also showed 629 571 micronemes and a polar ring. In the 4th instar larvae, the gamonts that As. taiwanensis completes the life cycle and remains infectious 630 572 (sometimes syzygies and gametocysts) are located either in the in O. j. japonicus (Erthal et al., 2012). The low host specificity of sev- 631 573 ectoperitrophic space (in younger larvae) or in the lumen of the eral other mosquito ascogregarines was shown too: As. barretti 632 574 intestine (in older larvae, after defecation prior to pupation). In developed in Ae. geniculatus (Rowton and Munstermann, 1984) 633 575 blood-fed females, the gametocysts attach to the accessory glands, and Ae. hendersoni (Copeland and Craig, 1992), and Spencer and 634 576 and the oocysts are injected into their lumen. In the males and Olson (1982) were able to infect Ae. epactius with As. culicis. 635 577 females fed exclusively on sugar, only gamonts were found Contrary to the above presented information, several authors 636 578 (Lantova and Volf, 2012). On average, oocysts measure showed that mosquito ascogregarines are fairly host specific. Dur- 637 579 9.6 Â 6.7 lm, gamonts measure 114.6 lm, and gametocysts are ing cross-infections of Ae. sierrensis with As. culicis and Ae. aegypti 638 580 128.2 lm in diameter (Lantova et al., 2010). The number of grega- with As. clarki, neither of the two gregarines produced oocysts in 639 581 rine parasites able to develop in a sand fly is limited and not corre- their unnatural hosts (Sanders and Poinar, 1973). Lien and 640 582 sponsive to the infection dose; in the 4th instar larvae, a ten-time Levine (1980) used oocysts of As. armigerei, As. culicis, As. lanyuen- 641 583 higher infection dose led to roughly 10 times more gamonts, while sis and As. taiwanensis to infect Ae. aegypti, Ae. albopictus, Ae. alca- 642 584 in adults, the intensity of infection achieved by a ten-time higher sidi and Ar. subalbatus. Even though the trophozoites of all 643 585 infection dose was only doubled (Lantova et al., 2011). The authors ascogregarines were recorded in all but one mosquito species 644 586 suggest that this decrease in gregarine numbers in adult sand flies (with lower infection rates), the oocysts were, apart from their 645 587 shows that the pupal stage is the most critical period for the sur- natural hosts, recovered only from Ae. alcasidi for As. taiwanensis 646 588 vival of Ps. sergenti. and As. armigerei. 647 In general, the host specificity studies of mosquito ascogrega- 648 589 4.6. Ps. tobbi rines gave contradictory results; some authors found these para- 649 sites to be fairly host specific, while others did not, and this 650 590 Ps. tobbi was described from Phlebotomus tobbi Adler, Theodor applies, in some cases when assessing a single species. For example, 651 591 and Lourie, 1930 laboratory-reared colony originating from Turkey the results of Lien and Levine (1980) suggesting high host specific- 652 Please cite this article in press as: Lantova, L., Volf, P. Mosquito and sand fly gregarines of the genus Ascogregarina and Psychodiella (Apicomplexa: Eugre- garinorida, Aseptatorina) – Overview of their taxonomy, life cycle, host specificity and pathogenicity. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/ j.meegid.2014.04.021 MEEGID 1944 No. of Pages 12, Model 5G 8 May 2014

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653 ity of As. lanyuensis (see above) contradict those of Jacques and Beier 6. Pathogenicity 718 654 (1982), who showed that this parasite completed its life cycle in ten 655 experimentally infected mosquito species. In addition, the host One of the first records about mosquito ascogregarines being 719 656 specificity of As. geniculati is questionable; the gregarine was able pathogenic brought Barrett (1968); Ae. aegypti larvae and pupae 720 657 to develop oocysts in Ae. sierrensis, Ae. aegypti and Ae. triseriatus infected with As. culicis were stunted, and the author noticed 721 658 (Munstermann and Levine, 1983), while, according to Kramar increased mortality. Sulaiman (1992) also observed increased lar- 722 659 (1952), it could not infect Aedes communis, Aedes cantans and Culex val mortality (proportional to the infection intensity) and short- 723 660 pipiens. It is appropriate to point out the wide host specificity across ened larval development; however, As. culicis did not affect the 724 661 various host orders of the genus Ascogregarina, as, apart from nem- larval development, size, mortality, pupal weight or adult emer- 725 662 atoceran mosquitoes, two species were described from brachyceran gence of Ae. aegypti in a study of McCray et al. (1970). As. barretti 726 663 flies and one from fleas (see Section 3.11). reduced the female pupal weight, prolonged the development of 727 664 As the host specificity of some species of mosquito ascogrega- males (Beier, 1983) and increased the likelihood of pupal mortality 728 665 rines is unclear, methods that would differentiate between individ- (Siegel et al., 1992). Contrastingly, emergence success (Copeland 729 666 ual species were studied. Several morphological features, and Craig, 1992; Walker et al., 1987) or larval developmental time 730 667 particularly the character of pigmentation and shape, distinguish- (Walker et al., 1987) were not affected. In addition, Beier (1983) 731 668 ing between gamonts of As. culicis and As. taiwanensis were found and Copeland and Craig (1992) did not record any increase in the 732 669 (Reyes-Villanueva et al., 2001). Oocysts of As. armigerei, As. culicis, larval mortality of As. barretti-infected Ae. triseriatus. 733 670 As. taiwanensis and Ascogregarina sp. from O. j. japonicus were com- Unlike for the immature mosquito stages, ascogregarines do not 734 671 pared under the scanning electron microscope, showing that they seem to be very pathogenic to their adult hosts. As. barretti does 735 672 differ mainly in the length and structure of their surface not alter the size of males and females (Walker et al., 1987) or their 736 673 (Roychoudhury et al., 2007a). As. barretti and As. geniculati were survival (Beier, 1983); however, the wing length of both sexes is 737 674 differentiated by isoenzyme electrophoresis (Rowton and reduced (Siegel et al., 1992). As. taiwanensis has a little impact on 738 675 Munstermann, 1984); different migration rates were observed for adults of Ae. albopictus (Garcia et al., 1994), and As. culicis-infected 739 676 isocitrate dehydrogenase, lactate dehydrogenase and malate dehy- Ae. aegypti do not have decreased survival or fecundity (McCray 740 677 drogenase, and the authors find this method reliable for distin- et al., 1970). An interesting study of Reeves (2004) revealed that 741 678 guishing between the two ascogregarines. A species specific PCR the rearing water from As. taiwanensis-infected larvae of Ae. aegypti 742 679 method for As. culicis and As. taiwanensis based on amplification is more acceptable for ovipositing female mosquitoes than the 743 680 of ribosomal ITS1 and ITS2 regions was developed by Morales water from gregarine-free larvae. 744 681 et al. (2005); the PCR products differed by at least 100 bp. In addi- Parasitism by Ascogregarina affects the hosts to a larger degree 745 682 tion, the authors found a diagnostic PCR method for the presence of when they are bred in nutrient-deficient conditions; the develop- 746 683 ascogregarines in mosquitoes. mental time of mosquito females is prolonged, the mortality of lar- 747 684 In the sand fly gregarines, unlike for widely studied mosquito vae and blood-fed females is significantly increased, the size of 748 685 ascogregarines, there are only two studies evaluating host specific- females and males is significantly smaller and the females produce 749 686 ity of the genus Psychodiella and both demonstrated it to be strict. fewer eggs (Comiskey et al., 1999a, 1999b; Walker et al., 1987). On 750 687 Seven phlebotomine species were infected with oocysts of Ps. the other hand, the emergence rate, developmental time and the 751 688 chagasi: Ph. papatasi, Ph. argentipes, Phlebotomus perniciosus, size of males are not affected (Walker et al., 1987). With regard 752 689 Lutzomyia serrana, Lutzomyia abonnenci, Lutzomyia columbiana and to the crowding effect on the hosts, the sex specific pattern of mos- 753 690 L. longipalpis. In the Old World sand flies, the trophozoites, and no quito reactions to the infection with Ascogregarina was supported 754 691 other gregarine life stages, occurred only in the newly emerged by Tseng (2004). 755 692 adults of Ph. papatasi. In four New World species studied, the tro- The effect of gregarines on mosquitoes can be substantially 756 693 phozoites were found in the adults of L. columbiana, L. serrana and altered by coinfections of the hosts with other parasites. Fellous 757 694 L. longipalpis, while the oocysts were found only in the natural host and Koella (2010) demonstrated that when Ae. aegypti larvae are 758 695 L. longipalpis. Furthermore, different colonies of L. longipalpis varied infected with high doses of As. culicis and Vavraia culicis (Fungi: 759 696 in the susceptibility to Ps. chagasi (Wu and Tesh, 1989). The authors Microsporidia) under low food availability conditions, the develop- 760 697 argue that the gregarines from a number of New World sand flies ment of the host is delayed; however, the mortality of immature 761 698 identified as Ps. chagasi (see Section 4.3) represent several new host stages is affected only mildly, and the wing length is not 762 699 species. altered. Furthermore, Fellous and Koella (2009) brought evidence 763 700 Strict host specificity was revealed also for Ps. sergenti, which that the coinfection with two parasites does not affect only their 764 701 was able to fully develop and complete its life cycle only in its nat- host but also the ability of the parasites to produce infective stages, 765 702 ural host Ph. sergenti. The oocysts were not recorded in experimen- and that these effects are dependent on the infection doses of the 766 703 tally infected Ph. papatasi or Phlebotomus arabicus, and they were parasites and on the food availability. 767 704 found only in a single blood-fed Ph. tobbi female out of 76 exam- The level of pathogenicity of mosquito ascogregarines is not 768 705 ined (Lantova et al., 2010). Similarly, Ps. tobbi was rarely able to influenced only by the sex, coinfection or nutrients, but it can be 769 706 produce oocysts in Ph. perniciosus females, and it did not complete significantly greater when the gregarine is introduced to an unnat- 770 707 its life cycle in Ph. sergenti (Lantova et al., 2010). ural host. For example, As. taiwanensis significantly increases the 771 708 When observing the dynamics of the infections of unnatural mortality of its unnatural host Ae. taeniorhynchus, while the mor- 772 709 hosts by either mosquito or sand fly gregarines, we can imply that tality of Ae. albopictus is not altered (Garcia et al., 1994). Similarly, 773 710 the bottleneck for the gregarines is the pupal stage. For instance, As. barretti decreases the larval survival, emergence success and 774 711 Erthal et al. (2012) observed 95% of O. j. japonicus larvae infected female weight of unnatural Ae. hendersoni, while the natural host 775 712 with As. taiwanensis, while the prevalence in pupae and adults Ae. triseriatus is not influenced (Copeland and Craig, 1992). Further- 776 713 was only 39% and 30%, respectively. Similar situation in mosqui- more, not the natural host Ae. aegypti but unnatural host Ae. epac- 777 714 toes was recorded by Garcia et al. (1994). Lantova et al. (2010) tius is negatively affected by simultaneous As. culicis infection and 778 715 found out that the prevalence of Psychodiella gregarines in the lar- methoprene, and the mortality rates are significantly increased 779 716 vae of their artificial hosts was roughly four times higher than in with increased methoprene concentrations (Spencer and Olson, 780 717 the adults. 1982). 781

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782 The pathogenicity of ascogregarines to their dipteran hosts is infected sand fly males and females without a blood meal was also 848 783 likely caused by the direct negative impact on the tissues where significantly decreased. Contrastingly, Ps. sergenti had no effect on 849 784 the parasites develop. The intestinal epithelial cells of the mos- the mortality of blood-fed females or their fecundity; the infection 850 785 quito larvae, the site of infection of the intracellular sporozoites did not affect the number of ovipositing females or the number of 851 786 and trophozoites, have enlarged nuclei (Kramar, 1952), and some oviposited eggs. 852 787 of them are destroyed by the gregarines (Kramar, 1952; Sanders As mentioned above, the intracellular development of Ascogreg- 853 788 and Poinar, 1973). The Malpighian tubules of adults, the site of arina is probably the cause of pathogenicity in the mosquitoes. On 854 789 infection of extracellular trophozoites and subsequent develop- the other hand, most Psychodiella gregarines do not develop intra- 855 790 mental stages, are dilated (Wenyon, 1911), and their cells are dis- cellularly, with the exception of Ps. mackiei; therefore, Lantova 856 791 torted and damaged (Barrett, 1968; McCray et al., 1970; Sanders et al. (2011) suggest that the main cause of higher mortality of 857 792 and Poinar, 1973). The extent of the damage is proportional to pre-imaginal sand flies could be competition for nutrients and 858 793 the infection rate, and as few as eight to 25 gametocysts may energy between the parasite and its host. Furthermore, the zero 859 794 destroy one third of a single Malpighian tubule (Barrett, 1968). effect on blood-fed females indicates that the nutrition could have 860 795 Ascogregarines may play a role in the ability of mosquitoes to an impact on adult sand fly response to the Psychodiella infections; 861 796 invade new areas. When Ae. aegypti and Ae. albopictus coexist in the blood meal (as a more nutritious diet) enables the infected 862 797 the same habitat, the latter tends to replace Ae. aegypti (Dos females to overcome the negative effect of the parasite. 863 798 Passos and Tadei, 2008). Two complimentary facts connected to Several authors have discussed the usefulness of Psychodiella 864 799 the infection with Ascogregarina could explain this phenomenon. and Ascogregarina parasites in biological control; Barrett (1968) 865 800 (1) When a mosquito species is introduced to a new region, there and Sulaiman (1992) consider them potentially useful, while 866 801 is a period when it is not infected or infected with a low rate by Walker et al. (1987), Wu and Tesh (1989), Siegel et al. (1992) 867 802 ascogregarines; e.g., Blackmore et al. (1995) showed that parasit- and Tseng (2007) do not. Experiments of Lantova et al. (2011) 868 803 ism by As. taiwanensis in the newly introduced Ae. albopictus showed that Ps. sergenti is harmful to its host, and that the effects 869 804 becomes higher only after three years. (2) Newly introduced inva- can be influenced by environmental factors. The authors point out 870 805 sive gregarine-free mosquitoes have competitive advantage over that this effect is very important in the laboratory colonies, where 871 806 the naturally occurring mosquitoes compared to the invasive greg- the infection intensity is usually much higher than in natural con- 872 807 arine-infected ones; e.g., according to Aliabadi and Juliano (2002), ditions. However, the authors claim that the potential of Psychodi- 873 808 invasive gregarine-free Ae. albopictus larvae reduced the survivor- ella gregarines for use in biological control is limited by their strict 874 809 ship of Ae. triseriatus, while gregarine-infected Ae. albopictus larvae host specificity and the lack of knowledge about sand fly breeding 875 810 did not. When considering both above mentioned points, the sites. 876 811 period of lower gregarine infection shortly after the introduction 812 of a new mosquito species to the area may give it competitive 813 advantage over the naturally occurring mosquito species; therefore 7. Parasite-host coevolution 877 814 enable the expansion of the invasive species. 815 The ability of Ascogregarina-infected mosquitoes to transmit Certain characteristics of Psychodiella and Ascogregarina suggest 878 816 parasites and viruses has been also studied. Mourya et al. (2003) close relationship of the gregarines with their hosts. In ascogrega- 879 817 showed that Chikungunya virus (Togaviridae) could be vertically rines, these aspects were studied e.g., by Roychoudhury et al. 880 818 transmitted to Ae. aegypti through As. culicis oocysts. On the other (2007a) who showed that, considering the oocyst morphology 881 819 hand, Miller and DeFoliart (1979) did not demonstrate that con- and nucleotide alignment of SSU rDNA, As. taiwanensis, As. culicis 882 820 comitant infection of Ae. triseriatus with As. barretti and La Crosse and Ascogregarina sp. from O. j. japonicus are more similar than 883 821 virus (Bunyaviridae) increases the infection rate of the virus, or As. armigerei. The authors suggest that these specific relationships 884 822 that As. barretti is a mechanism for the virus dispersal. Studies eval- of ascogregarines could be attributed to the different taxonomic 885 823 uating the effects of As. taiwanensis on the development of Dirofil- positions of their natural hosts. Similarly, in Psychodiella, Lantova 886 824 aria immitis (Nematoda: Spirurira) have come to contradictory et al. (2010) observed that Ps. sergenti and Ps. tobbi are, based on 887 825 conclusions. Comiskey et al. (1999b) found that in high nutrient their morphology and SSU rDNA sequences, closer to each other 888 826 conditions, D. immitis infective rate in Ae. albopictus females coin- than to Ps. chagasi, which corresponds to the origin of their hosts; 889 827 fected with As. taiwanensis was significantly higher than in the Ps. chagasi parasitizes New World L. longipalpis, while the other two 890 828 females infected only with D. immitis. This suggest that As. taiwan- gregarines are found in Old World members of the genus 891 829 ensis increases the vector competence of Ae. albopictus for filariae. Phlebotomus. 892 830 Contrastingly, Beier (1983) did not observe any significant differ- Another feature suggesting close relationship of ascogregarines 893 831 ences in the number of infective D. immitis larvae between grega- and mosquitoes is the developmental synchrony of their life cycles. 894 832 rine-infected and uninfected mosquitoes. The stage transformation of As. taiwanensis is conditioned by Ae. 895 833 In contrast to the large number of studies evaluating the effects albopictus metamorphosis and accelerated by a molting hormone 896 834 of ascogregarines on mosquitoes, there are only two works dealing 20-hydroxyecdysone (Chen and Yang, 1996). Furthermore, Chen 897 835 with this topic in sand flies. Wu and Tesh (1989) evaluated the et al. (2013) observed apoptosis of As. taiwanensis trophozoites that 898 836 effect of Ps. chagasi on L. longipalpis. The parasite significantly did not migrate from the intestine to the Malpighian tubules of Ae. 899 837 decreased the survival of L. longipalpis females, and the difference albopictus, and it was speculated that the apoptosis could regulate 900 838 was evident from day eight of the experiment. On the 25th day, the intensity of the parasite infection, maintaining the optimal sur- 901 839 the survivorship for the control females was 77.1% and for the vival rate of both the parasite and the host. 902 840 gregarine-infected females only 49.5%. Ps. chagasi did not signifi- The hypothesis about the coevolution of Psychodiella gregarines 903 841 cantly affect the fecundity of its host (Wu and Tesh, 1989). and sand flies is supported by several unique features of Psychodiella 904 842 Lantova et al. (2011) studied the pathogenic effect of Ps. sergenti life cycle. The location of the gregarines in the ectoperitrophic space 905 843 on its natural host Ph. sergenti. The gregarine negatively affected of the larval intestine (Lantova and Volf, 2012) protects them from 906 844 the survival of immature sand fly stages, as fewer adults emerged being defecated, helping to sustain a certain level of infection dur- 907 845 from the infected eggs than did so from the control ones. This effect ing pupation. The injection of the oocysts into the accessory gland 908 846 was even more pronounced under stressful conditions provided by lumen facilitated by the host immune response (Warburg and 909 847 rearing the infected larvae in higher density. The survival of Ostrovska, 1989) is a unique mode of vertical transmission. The 910 Please cite this article in press as: Lantova, L., Volf, P. Mosquito and sand fly gregarines of the genus Ascogregarina and Psychodiella (Apicomplexa: Eugre- garinorida, Aseptatorina) – Overview of their taxonomy, life cycle, host specificity and pathogenicity. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/ j.meegid.2014.04.021 MEEGID 1944 No. of Pages 12, Model 5G 8 May 2014

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911 attachment of the oocysts to the egg surface is then related to the Albicocco, A.P., Vezzani, D., 2009. Further study on Ascogregarina culicis in 972 temperate Argentina: prevalence and intensity in Aedes aegypti larvae and 973 912 exochorion formation (Lantova and Volf, 2012), and it facilitates pupae. J. Invertebr. Pathol. 101, 210–214. 974 913 the vertical transmission of the parasite. Additionally, in Ps. sergenti, Aliabadi, B.W., Juliano, S.A., 2002. Escape from gregarine parasites affects the 975 914 three interesting aspects should be highlighted. This gregarine does competitive interactions of an invasive mosquito. Biol. Invasions 4, 283–297. 976 977 915 not develop sexually in males or unfed females, which is advanta- Andreadis, T.G., 2007. Microsporidian parasites of mosquitoes. J. Am. Mosq. Control Assoc. 23 (Suppl. 2), 3–29. 978 916 geous for the gregarines, as they only invest energy into the sexual Ayala, S.C., 1971. Gregarine infections in the California sandfly, Lutzomyia vexatrix 979 917 development where the vertical transmission is expected – in occidentis. J. Invertebr. Pathol. 17, 440–441. 980 981 918 blood-fed females (Lantova and Volf, 2012). Unfed females die later Ayala, S.C., 1973. The phlebotomine sandfly-protozoan parasite community of central California grasslands. Am. Midl. Nat. 89, 226–280. 982 919 than males (Lantova et al., 2011), which gives the gregarine another Barrett Jr., W.L., 1968. Damage caused by Lankesteria culicis (Ross) to Aedes aegypti 983 920 advantage; the longer the female lives, the higher chance they have (L.). Mosquito News 28, 441–444. 984 921 for a blood meal followed by the egg production and transmission of Becnel, J.J., White, S.E., 2007. Mosquito pathogenic viruses – the last 20 years. J. Am. 985 Mosq. Control Assoc. 23 (Suppl. 2), 36–49. 986 922 the parasite. Lastly, the gregarine does not affect the mortality or Beier, J.C., 1983. Effects of gregarine parasites on the development of Dirofilaria 987 923 the fecundity of blood-fed females (Lantova et al., 2011), which immitis in Aedes triseriatus (Diptera: Culicidae). J. Med. Entomol. 20, 70–75. 988 924 gives the gregarines a bigger chance of being transmitted to the Beier, J.C., Chadee, D.D., Charran, A., Comiskey, N.M., Wesson, D.M., 1995. Country- 989 wide prevalence of Ascogregarina culicis (Apicomplexa: Lecudinidae), a 990 925 offspring. protozoan parasite of Aedes aegypti in Trinidad, West Indies. J. Am. Mosq. 991 Control Assoc. 11, 419–423. 992 Bhatia, B.L., 1938. Protozoa: sporozoa. In: Seweli, R.B.S. (Ed.), The Fauna of British 993 926 8. Conclusions India. Taylor and Francis, London, pp. 1–497. 994 Blackmore, M.S., Scoles, G.A., Craig Jr., G.B., 1995. Parasitism of Aedes aegypti and Ae. 995 927 The studies on mosquito and sand fly gregarines revealed very albopictus (Diptera: Culicidae) by Ascogregarina spp. (Apicomplexa: 996 Lecudinidae) in Florida. J. Med. Entomol. 32, 847–852. 997 928 interesting facts considering their taxonomy, when originally one Brazil, R.P., Ryan, L., 1984. Nota sobre a infeccao de Lutzomyia evandroi (Diptera: 998 929 genus Ascogregarina was shown to contain actually two distinct Psychodidae) por Ascocystis chagasi (Adler and Mayrink, 1961) no estado do 999 930 genera. Moreover, the phylogenetic closeness of these gregarines maranhao. Mem. Inst. Oswaldo Cruz 79, 375–376. 1000 Brazil, B.G., Teixeira, P.M.F., Minafra Jr., C.A., Temeljkovitch, M., Almeida, E.C.C., 1001 931 to neogregarines (e.g., Carreno et al., 1999) raised the question Brazil, R.P., 2002. Infeccao natural de Lutzomyia cruzi (Mangabeira, 1938) 1002 932 about their classification into eugregarines and put in doubt gen- (Diptera: Psychodidae: Phlebotominae) por Ascogregarina chagasi (Adler and 1003 933 eral methods for defining new gregarine species. The pathogenicity Mayrink, 1964). Entomol. Vectores 9, 289–293. 1004 1005 934 but also the host specificity of Ascogregarina and Psychodiella gre- Carreno, R.A., Martin, D.S., Barta, J.R., 1999. Cryptosporidium is more closely related to the gregarines than to coccidia as shown by phylogenetic analysis of 1006 935 garines are very important characteristics. Both these groups are apicomplexan parasites inferred using small-subunit ribosomal RNA gene 1007 936 considerably host specific, and their pathogenic effects on their sequences. Parasitol. Res. 85, 899–904. 1008 1009 937 hosts depend on various internal and external conditions. There- Chen, W.J., 1999. The life cycle of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae). Parasitol. Today 15, 153–156. 1010 938 fore, although the authors generally lean towards them not being Chen, W.J., Fan-Chiang, M.H., 2001. Directed migration of Ascogregarina taiwanensis 1011 939 very useful in biological control, they need to be considered in (Apicomplexa: Lecudinidae) in its natural host Aedes albopictus (Diptera: 1012 940 the maintenance of mosquito and sand fly colonies, which is so Culicidae). J. Eukaryot. Microbiol. 48, 537–541. 1013 Chen, W.J., Yang, C.H., 1996. Developmental synchrony of Ascogregarina taiwanensis 1014 941 important for successful research of human pathogens. (Apicomplexa: Lecudinidae) within Aedes albopictus (Diptera: Culicidae). J. Med. 1015 942 The host specificity, life cycle and molecular characteristics of Entomol. 33, 212–215. 1016 943 Ascogregarina and Psychodiella support the hypothesis about long Chen, W.J., Wu, S.T., Chow, C.Y., Yang, C.H., 1997a. Sporogonic development of the 1017 gregarine Ascogregarina taiwanensis (Lien and Levine) (Apicomplexa: 1018 944 and strong coevolutionary association between these parasites Lecudinidae) in its natural host, Aedes albopictus (Skuse) (Diptera: Culicidae). 1019 945 and their insect hosts. Even thought they are probably not applica- J. Eukaryot. Microbiol. 44, 326–331. 1020 946 ble in biological control, this coevolutionary relationship suggests Chen, W.J., Chow, C.Y., Wu, S.T., 1997b. Ultrastructure of infection, development and 1021 gametocyst formation of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) 1022 947 that there could be an interesting and important connection in its mosquito host, Aedes albopictus (Diptera: Culicidae). J. Eukaryot. Microbiol. 1023 948 between the hosts, their gregarine parasites and the human patho- 44, 101–108. 1024 949 gens transmitted by the hosts. Therefore, we trust that the impor- Chen, W.J., Huang, C.G., Fan-Chiang, M.H., Liu, Y.H., Lee, Y.F., 2013. Apoptosis of 1025 1026 950 tance and remarkably interesting life features of these parasites Ascogregarina taiwanensis (Apicomplexa: Lecudinidae), which failed to migrate within its natural host. J. Exp. Biol. 216, 230–235. 1027 951 will inspire scientists in further studies, bringing new information Christophers, S.R., 1960. Parasites. In: Aedes aegypti (L.), The Yellow Fever 1028 952 e.g., about the possible effect of gregarines on the vector compe- Mosquito: Its Life History, Bionomics, and Structure. Cambridge University 1029 1030 953 tence of mosquitoes and sand flies. Press, London, pp. 63–77. Clark, T.B., 1980. Non-microsporidian protozoan pathogens of Culicidae 1031 (mosquitoes). Bull. World Health Organ. 58 (Suppl.), 47–52. 1032 954 Acknowledgments Coelho, M.V., Falcao, A.L., 1964. Aspects of the life-cycle of ‘‘Monocystis chagasi’’ 1033 Adler and Mayrink, 1961, in ‘‘Phlebotomus longipalpis’’. Rev. Bras. Biol. 24, 417– 1034 421. 1035 955 We would like to thank Prof. Jiri Vavra and the reviewers for crit- Comiskey, N.M., Lowrie Jr., R.C., Wesson, D.M., 1999a. Role of habitat components 1036 956 ical reading of the manuscript, Doc. Milena Svobodova and Doc. Jan on the dynamics of Aedes albopictus (Diptera: Culicidae) from New Orleans. J. 1037 Med. Entomol. 36, 313–320. 1038 957 Votypka for providing useful comments on the manuscript and Ema Comiskey, N.M., Lowrie Jr., R.C., Wesson, D.M., 1999b. Effect of nutrient levels and 1039 958 Klobusovska for assistance in the preparation of the artworks. Lucie Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) infections on the vector 1040 959 Lantova was supported by research projects of the Charles competence of Aedes albopictus (Diptera: Culicidae) for Dirofilaria immitis 1041 1042 960 University in Prague UNCE 204013 and PRVOUK P25/LF1/2. Petr (Filarioidea: Onchocercidae). J. Med. Entomol. 36, 55–61. Copeland, R.S., Craig Jr., G.B., 1992. Interspecific competition, parasitism, and 1043 961 Q2 Volf was supported by Ege Universitylji University Grant predation affect development of Aedes hendersoni and A. triseriatus (Diptera: 1044 962 2011-261504 EDENext, and the article is catalogued by the Culicidae) in artificial treeholes. Ann. Entomol. Soc. Am. 85, 154–163. 1045 1046 963 EDENext Steering Committee as EDENext 230. Dellape, M.E., Marti, G.A., Tranchida, M.C., Garcia, J.J., 2005. First record of Aedes aegypti (L.) 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