Systematic studies of Anopheles (Cellia) kochi (Diptera: Culicidae): morphology, cytogenetics, cross-mating experiments, molecular evidence and susceptibility level to infection with nocturnally subperiodic Brugia malayi

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Systematic studies of Anopheles (Cellia) kochi (Diptera: Culicidae): morphology, cytogenetics, cross-mating experiments, molecular evidence and susceptibility level to infection with nocturnally subperiodic Brugia malayi

Watchara Jatuwattana InvestigationData curationWriting original draft preparation , Atiporn Saeung ConceptualizationData curationFormal analysisWriting - original draft preparationWriting - review a Kritsana Taai , Wichai Srisuka , Sorawat Thongsahuan , Kittipat Aupalee , Petchaboon Poolphol , Kanchon Pusawang , Pradya Somboon Writing - review and editing , Wanchai Maleewong SupervisionFunding Acquisition PII: DOI: Reference:

S0001-706X(19)31537-2 https://doi.org/10.1016/j.actatropica.2019.105300 ACTROP 105300

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

4 November 2019 12 December 2019 12 December 2019

Please cite this article as: Watchara Jatuwattana InvestigationData curationWriting original draft preparation , Atiporn Saeung ConceptualizationData curationFormal analysisWriting - original draft preparationWriting - review a Kritsana Taai , Wichai Srisuka , Sorawat Thongsahuan , Kittipat Aupalee , Petchaboon Poolphol , Kanchon Pusawang , Pradya Somboon Writing - review and editing , Wanchai Maleewong SupervisionFunding Acquisition , Systematic studies of Anopheles (Cellia) kochi (Diptera: Culicidae): morphology, cytogenetics, cross-mating experiments, molecular evidence and susceptibility level to infection with nocturnally subperiodic Brugia malayi, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105300

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Highlights

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This study is the first to report new metaphase karyotypes of Anopheles kochi in Thailand.

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This is the first report to provide clear evidence that two different cytological forms of An. kochi are conspecific by using multidisciplinary approaches.

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Female An. kochi bear five types of antennal sensilla, identified by SEM.

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The cibarial armature of An. kochi is an important factor that acts as the first line of defense against filarial infection by inflicting lethal macerations on microfilariae.

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Systematic studies of Anopheles (Cellia) kochi (Diptera: Culicidae): morphology, cytogenetics, cross-mating experiments, molecular evidence and susceptibility level to infection with nocturnally subperiodic Brugia malayi Watchara Jatuwattanaa,b, Atiporn Saeungb*, Kritsana Taaic, Wichai Srisukad, Sorawat Thongsahuane, Kittipat Aupaleeb, Petchaboon Poolpholf, Kanchon Pusawangb, Pradya Somboonb and Wanchai Maleewongg a

Graduate Master’s Degree Program in Parasitology, Faculty of Medicine, Chiang Mai

University, Chiang Mai 50200, Thailand b

Center of Insect Vector Study, Department of Parasitology, Faculty of Medicine,

Chiang Mai University, Chiang Mai 50200, Thailand c

Faculty of Veterinary Medicine, Western University, Kanchanaburi 71170, Thailand

d

Entomology Section, Queen Sirikit Botanic Garden, Chiang Mai 50180, Thailand

e

Faculty of Veterinary Science, Prince of Songkla University, Songkhla 90110, Thailand

f

The Office of Disease Prevention and Control Region 10th, Ubon Ratchathani, 34000,

Thailand g

Department of Parasitology, Research and Diagnostic Center for Emerging Infectious

Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand *Corresponding author: [email protected] (A .Saeung) E-mail address:

[email protected] (W. Jatuwattana) [email protected] (A. Saeung) [email protected] (K. Taai) [email protected] (W. Srisuka) [email protected] (S. Thongsahuan) [email protected] (K. Aupalee) [email protected] (P. Poolphol) [email protected] (K. Pusawang) [email protected] (P. Somboon) [email protected] (W. Maleewong)

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ABSTRACT Anopheles kochi Dӧnitz (Diptera: Culicidae) is a malaria vector in some countries in South and Southeast Asia. This is the first report to provide clear evidence that two different cytological forms of An. kochi are conspecific based on systematic studies. Two karyotypic forms, i.e., Form A (X1, X2, Y1) and a novel Form B (X1, X2, Y2) were obtained from a total of 15 iso-female lines collected from five provinces in Thailand. Form A was common in all provinces, whereas Form B was restricted to Ubon Ratchathani province. This study determined whether the two karyotypic variants of An. kochi exist as a single or cryptic species by performing cross-mating experiments in association with the sequencing of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA), and cytochrome c oxidase subunit I (COI) of mitochondrial DNA (mtDNA). Cross-mating experiments between the two karyotypic forms revealed genetic compatibility by providing viable progenies through F2 generations. The two forms showed a high sequence similarity of those two DNA regions (average genetic distances: ITS2 = 0.002-0.005, COI = 0.000-0.009). The phylogenetic trees based on ITS2 and COI sequences also supported that four strains (from Bhutan, Cambodia, Indonesia, and Thailand) were all of the same species. Five sensilla type house on the antennae of female An. kochi were observed under scanning electron microscopy (SEM). In addition, this study found that An. kochi was a refractory vector, revealed by 0% susceptibility rates to infection with nocturnally subperiodic Brugia malayi. The cibarial armature was a resistant mechanism, as it killed the microfilariae in the foregut before they penetrated into the developmental site. Keywords: Anopheles kochi, Antennal sensilla, Metaphase karyotype, Cross-mating experiments, second internal transcribed spacer, cytochrome c oxidase subunit I, Brugia malayi

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1. Introduction

To date, a total of 476 formally recognized species of Anopheles have been reported worldwide (Harbach, 2019). Anopheles (Cellia) kochi Dӧnitz is one of 77 species occurring in Thailand (Rattanarithikul et al., 2006). It belongs to the Kochi group of the Neomyzomyia Series. It is distributed widely in the Oriental region, from India, Nepal, Bhutan, South China, Myanmar, Thailand towards the Philippines and Indonesia (Christophers, 1933; Bonne-Wepster and Swellengrebel, 1953; Reid, 1968; Darsie and Pradhan, 1990; Rattanarithikul et al., 2006; Elyazar et al., 2013; Bhattacharyya et al., 2014; Namgay et al., 2018). It bites both humans and cattle with a major biting peak during the first half of the night (18:00-20:00 h) (Pinontoan et al., 2017). Anopheles kochi is a malaria vector in Sumatra, Indonesia (Elyazar et al., 2013). Anopheles kochi plays a significant role in malaria transmission, as observed on the Bangladesh-Indian border, with CSP-ELISAs and Plasmodium DNA finding positive results for P. vivax (Pv-210, Pv-247) and P. falciparum (Al-Amin et al., 2015; Alam et al., 2012; Bashar et al., 2013; Dutta et al., 1993). It is a potential vector of human malarial parasites in Thailand, with its susceptibility to both P. falciparum and P. vivax (Somboon et al., 1994). Using CSP-ELISAs, P. vivax infections were detected in six of 336 (infection rates = 1.78) samples of An. kochi in South Halmahera, in the northern Maluku islands (St Laurent et al., 2017). Lien et al. (1991) reported that An. kochi was susceptible to infection with three human Plasmodium spp., P. falciparum, P. vivax and P. malariae, in Taiwan. The report of Japanese Encephalitis (JE) virus in An. kochi was observed in Indonesia (Garjito et al., 2018). However, the susceptibility level of An. kochi to filarial parasites was not known until the present study. According to cytogenetic studies, Baimai et al. (1996) reported two types of X chromosome (X1, X2) and one type of large telocentric Y chromosome of An. kochi in northern and southern regions of Thailand. Recently, our preliminary study revealed a novel form of small telocentric Y chromosome, which is hereafter referred to Form B and that of Baimai et al. (1996) as Form A. Whether Form B reflects interspecific variation was investigated in the current study by using cross-mating experiments and phylogenetic analyses of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) and cytochrome c oxidase subunit I (COI) of mitochondrial DNA

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(mtDNA) sequences. Furthermore, the ultrastructure of the antennal sensilla of An. kochi was examined. The susceptibility levels of An. kochi to infection with nocturnally subperiodic (NSP) Brugia malayi under laboratory conditions, and the factor affecting the susceptibility of mosquitoes to the filarial parasite were also reported herein for the first time.

2. Materials and methods

2.1 Field collections and establishment of isoline colonies

Wild-caught, fully engorged An. kochi female mosquitoes were collected from cow-baited traps at five locations in three regions of Thailand: 1) northern region (Mae Taeng district, Chiang Mai province and Phu Phiang district, Nan province); 2) northeastern region (Na Chaluai district, Ubon Ratchathani province); and 3) southern region (Thung Kha Ngok district, Phang Nga province and Wang Wiset district, Trang province). Additional specimens from other countries in Southeast Asia, including Bhutan, Cambodia, and Indonesia, were included for DNA analysis. The adult mosquitoes were identified to species using the taxonomic key of Rattanarithikul et al. (2006) and Somboon and Rattanarithikul (2013). A total of 15 isolines were established successfully and maintained in the insectary at the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand, using the techniques described by Choochote and Saeung (2013).

2.2 Metaphase chromosome preparation

Ten brains of early fourth-instar larvae derived from F1- and/or F2-progenies of each isoline of An. kochi were used for metaphase chromosomes, using the method of Saeung et al. (2007). The larvae were incubated in 1% solution of dried Gloriosa superba L. seed powder for 2 h at room temperature. The dissected heads were incubated and left in a 1% sodium citrate solution on a siliconized slide for 10 min. The brains were removed carefully by dissecting needles and transferred to Carnoy’s fixative solution on a siliconized slide for 2 min. Then, a drop of 60% acetic acid was

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added, and the organs were torn and mixed well with dissecting needles. A drop of cell suspension was pipetted and transferred by smearing onto a microscopic slide on a warming plate. Dried samples were stained with 10% Giemsa in phosphate buffer pH 7.2, rinsed with deionized water, air-dried at room temperature and mounted in Permount. Identification of karyotypic forms followed Baimai et al. (1996).

2.3 Cross-mating experiments

Five laboratory-raised isolines of An. kochi were selected arbitrarily from the 15 isoline colonies, which were representative of two karyotypic forms, i.e., Form A (Cm1A, Nn-2A, Tr-2A, and Pg-3A) and Form B (Ur-3B) (Table 1). These isolines were used for the cross-mating experiments to determine genetic compatibility by following the procedures reported by Saeung et al. (2007). Briefly, adult females and males emerged from pupae that had been sexed and placed individually in test tubes. Artificial mating between males and blood-fed virgin females was performed using the technique of Ow Yang et al. (1963). The gravid females were allowed to oviposit individually in a small plastic cup, and the eggs were counted and placed in hatching pans. The embryonation, hatching, pupation, emergence rates, sex ratio, and morphology of testes and ovaries of F1 hybrids were observed and recorded. Then, the fertility and viability of the F2 hybrids produced from the F1 hybrids were also determined using the methods as above. After completing the cross-mating experiments, the isoline of each form was pooled in order to establish Form A and Form B colonies, which were used for further studies on the ultrastructure of antennal sensilla under scanning electron microscope (SEM) and the susceptibility level to infection with B. malayi (see below).

2.4 DNA extraction and amplification

Genomic DNA was extracted from individual adult female mosquitoes (n = 24) using the PureLink ® Genomic DNA Mini Kit (Invitrogen, USA). The ITS2 and COI regions were amplified using the universal forward and reverse primers as described by Saeung et al. (2007, 2008). Polymerase Chain Reaction (PCR) was carried out using 20

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µl volumes containing 0.5 U Taq DNA polymerase, 3 mM of MgCl2, 0.2 mM of each dNTP, 0.25 µM of each primer, and 2 µl of the extracted DNA. The conditions for ITS2 amplification consisted of initial denaturation at 95oC for 1 min, 35 cycles at 95oC for 30 sec, 55oC for 30 sec, and 72oC for 1 min, and a final extension at 72oC for 10 min. The amplification profile of COI comprised initial denaturation at 94oC for 2 min, 35 cycles at 94oC for 30 sec, 45oC for 30 sec, and 72oC for 30 sec, and a final extension at 72oC for 5 min. The amplified products were electrophoresed on 1.5% agarose gel. The DNA sequencing was performed at Macrogen (Seoul, Korea). Newly determined sequences were deposited in the GenBank nucleotide sequence database (Table 1). The sequences obtained from this study were compared with deposited sequences available in GenBank.

2.5 Sequence alignment and phylogenetic analysis

Sequences of ITS2 and COI were aligned using the CLUSTALW multiple alignment program (Thompson et al., 1994) and the alignments were then edited manually. Gap sites were excluded from the following analysis. The genetic distances were calculated with the Kimura two-parameter (K2P) method (Kimura, 1980). Phylogenetic relationships between sequences of An. kochi were inferred using maximum likelihood (ML) and neighbor-joining (NJ) methods (Saitou and Nei, 1987). Bootstrap support for ML and NJ trees were analyzed using the MEGA version 7.0 program (Kumar et al., 2016). For ML trees, the best-fit model for ITS2 and COI sequences were T92 and T92 + Gamma, respectively.

2.6 Scanning electron microscopy (SEM)

The protocol for SEM used the method described by Taai et al. (2017). Briefly, 30 heads of five-day-old female An. kochi were excised under a stereo microscope and rinsed three times in phosphate buffer (pH 7.4) to remove surface debris. The heads were then dehydrated through an ethanol series of 35%, 70%, 80% (10 min, two changes) and 95% (15 min, two changes), followed by absolute ethanol (10 min, two

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changes) and dried in a critical point dryer. The antennae were dissected from the head capsule under a stereomicroscope, mounted on aluminum stubs with double-sided carbon adhesive tape and sputter-coated with gold. Sensilla were observed and photographed by using a JEOL-JSM6610LV scanning electron microscope (JEOL, Japan) at the Medical Science Research Equipment Center, Faculty of Medicine, Chiang Mai University, Thailand. Identification of sensilla types followed the terminology described by Hempolchom et al. (2017) and Taai et al. (2017).

2.7 Susceptibility of An. kochi to infection with NSP B. malayi

Five-day-old Aedes togoi adult females (control) and laboratory-raised strains of An. kochi (fasted for 12 h) were allowed to feed simultaneously on blood containing microfilariae (mf) of a NSP B. malayi strain (Choochote et al., 1986) (microfilariae density = 261 and 281 mf/20 µl in experiments 1 and 2, respectively) using the artificial membrane feeding technique (Saeung and Choochote, 2013). After the blood meal, at least five fully engorged mosquitoes were dissected at different time points (5 min, 1 h, 12 h and 96 h) in order to determine the migration of microfilariae. The midgut was pulled out carefully and transferred to a new glass slide, then opened to make a thick blood film, dried out, dehemoglobinized, fixed with absolute methanol, and stained with 10% Giemsa stain in phosphate buffer pH 7.2. The development and morphology of microfilariae in each sample were examined under a compound microscope. Photographs of the microfilariae were taken using a BX53 microscope and Olympus DP72 camera with cellSens standard imaging software (Olympus, Japan). Fourteen days after infection, all infected mosquitoes were dissected in normal saline solution. The number of mosquitoes with at least one third-stage larva (L3) in any parts of the body (head, thorax or abdomen) (n = 30 female mosquitoes) was recorded for determining the infection rates and parasite loads.

2.8 Ethical clearance The protocol used in this study was approved by the Research Ethics Committee (Permit No. 16/2562), Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.

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3. Results 3.1 Metaphase karyotype

The results of investigation on 15 An. kochi isolines revealed two types of X chromosomes, a telocentric X1 and a large telocentric X2, as reported by Baimai et al. (1996) (Fig. 1A, B). The telocentric X2 was slightly longer than the X1 chromosome, due to the presence of centromeric heterochromatin. Interestingly, there are two types of Y chromosome: a large telocentric Y chromosome as reported by Baimai et al., (1996) and a new small telocentric Y chromosome, which is referred to Y1 and Y2, respectively, in the current study (Fig. 1A, B). The large telocentric Y1 chromosome was slightly shorter than the X1 chromosome, as reported by Baimai et al. (1996). The diagrammatic representations of the metaphase karyotypes of An. kochi are shown in Fig. 1C, D. Based on uniquely different characteristics of the Y chromosome, they were designated as Form A (X1, X2, Y1) and Form B (X1, X2, Y2). Form A was found in several provinces of Thailand, including, Chiang Mai, Nan, Phang Nga and Trang, but Form B was found in only Ubon Ratchathani province, northeastern Thailand.

3.2 Cross-mating experiments

All crosses were compatible genetically and yielded viable progenies through F2 generations. The embryonation, hatchability, pupation, emergence rates, and ratio of adult females/males of parental, reciprocal, and F1-hybrid crosses were presented in Table 2.

3.3 DNA sequences and phylogenetic analysis

The ITS2 and COI sequences were obtained from 15, 3, 3, and 3 individual An. kochi isolines collected from Thailand, Bhutan, Cambodia, and Indonesia, respectively (Table 1). They all showed the same lengths of ITS2 (574 bp) and COI (658 bp). Based on ITS2 and COI sequences, the average genetic distances within and between the two karyotypic forms (Forms A and B) of An. kochi showed a very low level in two DNA

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regions (ITS2 = 0.002-0.005, COI = 0.000-0.009). The ML and NJ trees yielded a similar tree topology, showing a monophyletic clade of Thai An. kochi (Forms A and B) (Fig. 2, 3). Likewise, An. kochi from other countries were clustered within the same clade (average genetic distances = 0.000-0.013), and well separated from the outgroups (An. cracens, An. dirus and An. tessellatus). One published ITS2 (GenBank accession numbers EU650424) (Fig. 2) and five published COI sequences (GenBank accession numbers KF564706, KF564707, KF564708, JQ728290, JQ728292) (Fig. 3), which were identified previously as An. kochi from China and Singapore, were also placed within the same clade as the specimens in this study.

3.4 Morphology of antennal sensilla of female An. kochi 3.4.1 Genaral morphology of antennal sensilla

The antennae of An. kochi consist of three parts: scape, pedicel, and flagellum (Figs. 4A and 4B). The scape (Sc) is collar-shaped and attached behind the pedicel (Pe), which is a cup-shaped organ containing Johnston’s organ and acts as the junction of flagellum. Each flagellum is divided into 13 flagellomeres (Fig. 4A). The entire surface of the scape, pedicel, and first flagellomere is covered densely with aculeae (ac, microtrichium-like spicules) (Figs. 4B and 5). Some scales are found on the pedicel and first flagellomere. Five types of sensilla are borned on the female antennae of An. kochi: ampullacea (sa), sensilla chaetica (ch), sensilla trichodea (tc), sensilla basiconica (sb) and sensilla coeloconica (co).

3.4.2 Different types of sensilla observed on antennae

Sensilla ampullacea (pegs in tubes) (sa) are small peg-like or slit-like organs of about 1 µm in diameter (Fig. 5, 6A). This sensilla type is only found on the first flagellomere. Sensilla chaetica (ch) are long, thick-walled setae (bristles), with their base inserted into a socket and longitudinal grooves presented on the cuticular surface. There are two subtypes: large (lch) and small (sch), based on their size (Fig. 5). The lch is

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found mostly at the base of flagellomeres 2-13, whereas sch usually occurs on the distal ends. Both subtypes also occur on the surface of the first flagellomere (Fig. 5). Sensilla trichodea (tc) are hair-like sensory structures and not set into a socket. They are the most numerous sensilla type, which presents on the entire flagellum. Sensilla trichodea have three subtypes: long sharp-tipped trichodea (ltc), short sharptipped trichodea (stc) and blunt-tipped trichodea (btc) (Fig. 5). The long sharp-tipped trichodea have a smooth surface that gradually tapers distally to a tip. Their number increases from the proximal to the distal ends of the flagellomeres. The short sharptipped trichodea are bent slightly and fewer in number than the former type (Fig. 5). Blunt-tipped trichodea are shorter in length, with rounded tips that are nearly as wide as the base diameter (Fig. 5). They also occur in fewer numbers than the sharp-tipped trichodea. Sensilla basiconica (grooved pegs) (sb) have curved peg-like or thorn-shaped hairs with 10-12 longitudinal grooves on their surfaces, and are raised on small prominences that lack sockets (Fig. 5, 6B). They are observed on the entire flagellum. Sensilla coeloconica (pitted pegs) (co) have thick-walled sensilla. There are two subtypes based on the shape of these sensilla, large and small (Fig. 5). The large sensilla coeloconica (lco) are characterized by their pegs, which may or may not project from the floor of shallow depressions through circular openings at the cuticle surface. They have approximately 10-12 deep longitudinal grooves on their walls (Fig. 5, 6C). This type of sensilla is observed on flagellomeres 1-12 with less abundance than sensilla trichodea or sensilla basiconica. The small sensilla coeloconica (sco) have a tiny invisible peg set at the bottom of a shallow pit, with a much smaller cuticular opening on the surface than that on large coeloconica. Sensilla coeloconica occur on the first flagellomere and tip of flagellomere 13 (Fig. 6D, E).

3.5 Susceptibility of An. kochi to infection with NSP B. malayi

Details of the infective rate (IR) and parasite load of Ae. togoi (control) and An. kochi 14 days after feeding on blood containing B. malayi mf are shown in Table 3. The IR and average number of L3 larvae per infected Ae. togoi mosquito (AL3) were

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calculated [experiment 1 (IR = 46.67%, AL3 = 1-14) and experiment 2 (IR = 50%, AL3 = 1-40)]. No infective larva was found in any parts of An. kochi, and the IR was 0%. Of the 4 time points (5 min, 1 h, 12 h, and 96 h), the mean number of mf found in the midgut was 21.40, 8.0, 3.0 and 1.5, respectively, for Ae. togoi, and 6.0, 4.67, 1.67, and 1.5, respectively, for An. kochi. The highest number of mf was observed at 5 min in the midgut of Ae. togoi, and it declined gradually after the mf migrated to the thoracic musculature. The mf recovered from the midgut of Ae. togoi at 5 min (Fig. 7A), 1 h (Fig. 7C) and 12 h (Fig. 7E), showing normal morphology (intact sheath, cuticle and column nuclei). In addition, normal first-stage larvae (L1) were found in thoracic muscle fibres at 96 h (Fig. 7G). In contrast, all of the mf had died in the midgut of An. kochi, and all of them were broken or damaged as the nuclei were seen to protrude through a ruptured cuticle and sheath (Fig. 7D, F). The broken mf were seen apparently within 5 min (Fig. 7B). According to the mf morphology found in this study, it was determined whether the cibarial armature played the role of a barrier to microfilarial development in the mosquitoes. The cibarial armature was found in An. kochi, but not in Ae. togoi. Anopheles kochi has well-developed cibarial armatures consisting of 12 large sharp teeth, of which two lateral ones are small or not fully developed. The number of deep clefts for each tooth varied from 1 to 3. The pediment (Pd) of each tooth had two laterally small teeth (Fig. 7H).

4. Discussion It has long been known that the evolution of species complexes in anopheline vectors leads to difficulty in precisely identifying sibling species (isomorphic species) and/or subspecies (cytological races) members that possess identical morphology or minimal morphological distinction (Choochote and Saeung, 2013). The exact identification of Anopheles vector species is the backbone for planning an effective control program. Some previous studies have revealed that differences in the mitotic chromosome can be utilized for species identification among closely related Anopheles malaria species (Baimai et al., 1987, 1988). Differences in karyotypes must reflect species distinction in many Anopheles species, but in some, such differences are proved to be intra-specific variations (WHO, 2007). In addition, at least 1 or 2 traditional

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techniques (e.g. isoenzymes and polytene chromosomes) have so far been used for the recognition of sibling species and/or subspecies members. However, several problems have been raised regarding these techniques. To overcome these problems, Choochote and Saeung (2013) developed robust systematic techniques that successfully identified three new species within the Barbirostris Complex, that were further specifically differentiated using a multiplex PCR (Taai and Harbach, 2015; Brosseau et al. 2019). Although An. kochi is found commonly throughout Thailand and has medical importance, only one report regarding the metaphase karyotype of An. kochi has been made in Thailand since 1996, when Baimai et al. stated that the metaphase karyotype of An. kochi consists of two types of X chromosome and one type of Y. In this study, An. kochi isolines from five allopatric locations in Thailand revealed two types of X and Y chromosomes, which were designated as Form A and Form B, based on the uniquely distinct characteristics of Y chromosomes. This study hypothesized that the two karyotypic variants of An. kochi may have evolved into cryptic species. Therefore, systematic techniques (Choochote and Saeung, 2013), including morphological taxonomy, cross-mating experiments, cytogenetics (characteristics of metaphase karyotypes), and molecular investigation, were applied to An. kochi in order to clarify the hypothesis in this study. However, the results of the current study provided clear evidence that both Form A and Form B are conspecific. The findings in this study were consistent with those of a previous one by Baimai et al. (1996), but the novel metaphase karyotype, Form B (a small telocentric Y2 chromosome), was first described herein. Baimai et al. (1984, 1988, 1993) suggested that the quantitative differences in the heterochromatin of mitotic chromosomes could be used as a genetic marker for further identification of cryptic (isomorphic) or closely related species, such as the cytogenetic population studies of the Dirus complex and Maculatus Group. It was interesting to note that the novel Form B was found in the specifically karyotypic form in Ubon Ratchathani province, northeastern Thailand, whereas the remaining isolines collected from northern and southern regions showed similar karyotypic forms (Form A). The results of this study are in accordance with Songsawatkiat et al. (2014) and Saeung et al. (2014). Songsawatkiat et al. (2014) demonstrated that An. nitidus Forms D and E of the Hyrcanus Group were detected only in Ubon Ratchathani province, whereas, Forms A, B, and C were found from three

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isolines in Phang Nga province. Likewise, Saeung et al. (2014) reported that An. crawfordi Forms A and B of the same Hyrcanus Group appear to be shared in all locations of both Thailand and Cambodia, whereas, Forms C and D are confined to Trang province, southern Thailand. This phenomenon seemed to associate with specific microhabitats that favored karyotypic forms of An. kochi. However, additional isolines of An. kochi collected from other locations are needed before it can be concluded that ecological diversity is responsible for this phenomenon. Cross-mating experiments among the two karyotypic forms of An. kochi clearly revealed that they are genetically compatible. They yielded viable progenies through F 2 generations. Thus, the results of this study indicated that the two karyotypic forms are of the same species. These results agree with previous cross-mating experiments among the karyotypic forms of other anopheline species, for example, An. vagus Forms A and B (Choochote et al., 2002), An. sinensis Forms A and B (Choochote et al., 1998, Park et al., 2008), An. aconitus Forms B and C (Junkum et al., 2005), An. dissidens Forms A, B and C and An. saeungae Forms A and B (Saeung et al. 2007, Suwannamit et al., 2009), An. wejchoochotei Forms B, E and F (Thongsahuan et al., 2009), An. peditaeniatus Forms A and B (Choochote, 2011, Saeung et al., 2012), An. nigerrimus Forms A and B (Songsawatkiat et al., 2013), An. nitidus Forms A and B (Songsawatkiat et al., 2014), An. argyropus Forms A and B and An. pursati Forms A, B and C (Thongsahuan et al., 2014). Furthermore, the monophyletic trees and very low K2P genetic distances of rDNA ITS2 and mtDNA COI sequences between two forms, as well as among four other strains (Bhutan, Cambodia, Indonesia and Thailand) of An. kochi, are good supportive evidence that confirms the existence of a single cosmopolitan species. Basically, mosquitoes mainly rely on the sense of smell to find a mate, nectar, blood and oviposition sites. Understanding the mechanisms of mosquito olfaction and odor coding is crucial in developing measures based on behavioral disruption for mosquito control (Qui and van Loon, 2010). The antennae of adult mosquitoes carry numerous sensory structures called sensilla, which are the physical sites for odor detection. The behavioral responses to volatile cues, including a female mosquito finding a host, are a critical factor in the ability of mosquitoes to transmit pathogens responsible of diseases (Ibrahim et al., 2018). Even though several studies have investigated the antennae of medically important Anopheles mosquitoes to date

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(Hempolchom et al., 2017; Taai et al., 2017, 2019), data are lacking on the morphology of this organ in An. kochi. Hence, this study reports the first SEM investigation on morphology of the antennae of female An. kochi. It provides a database of this malaria vector, which is useful for further development of odorant-based methods for reducing disease transmission by preventing mosquito-human interactions. The results of this study show that the antennae of An. kochi are equipped with five different types of sensilla, namely sensilla ampullacea, sensilla chaetica, sensilla trichodea, sensilla basiconica and sensilla coeloconica, which are similar morphologically to those described in many other Anopheles species, such as, An. gambiae s.s. and An. quadriannulatus (Pitts and Zwiebel, 2006), An. argyropus, An. crawfordi, An. nigerrimus, An. nitidus, An. lesteri, An. peditaeniatus, An. pursati, and An. sinensis (Hempolchom et al., 2017), An. minumus and An. harrisoni (Taai et al., 2017), and An. dirus and An. cracens (Taai et al. 2019). Sensilla ampullacea are sensitive to thermal and moisture stimuli (Sutcliffe, 1994). It has been suggested that the function of sensilla chaetica and sensilla trichodea is that of mechanoreceptors (Hill et al., 2009). Broek and Otter (1999) reported that olfactory receptor neurons (ORNs) on sensilla trichodea respond to carboxylic acids and 1-octen-3-ol. Sensilla basiconica act as olfactory sensilla (Sutcliffe, 1994) or chemoreceptors, while sensilla coeloconica have been putatively classified as hygro- and thermo-receptors (Ibrahim et al., 2018). Anopheles kochi is refractory to NSP B. malayi comparing its infective rate with that of Ae. togoi, which had proved to be an efficient laboratory vector (Saeung and Choochote, 2013). Furthermore, observations on the migration of B. malayi mf in Ae. togoi and An. kochi revealed the relationship between the presence of cibarial armatures and the numbers of broken and dead mf in the midgut by Giemsa staining infected mosquitoes at four different time points. The cibarial armature is a set of teeth that projects into the posterior lumen of the cibarium, and acts as the first line of defense against filarial infection by inflicting lethal macerations on microfilariae (Choochote et al., 1987). Denham and McGreevy (1977) suggested that the degree of damage depends on the size and structure of the armatures. Well-developed teeth observed in the cibarial armature of An. kochi killed mf in the midgut, which is in accordance with those observed in other mosquito species, such as, four species members of the Dirus Complex (An. dirus, An. baimaii, An. cracens and An. scanloni) (Choochote et al.,

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1987; Somboon et al., 2009), two species members of the Gambiae Complex (An. gambiae s.s. and An. arabiensis), An. farauti s.s (McGreevy et al., 1978), and An. vagus (Choochote et al., 1987). In contrast, no broken mf were observed in the midgut of Ae. togoi, because this species lacks the cibarial armature, which is similar to observations in the study of Choochote et al. (1987). This study found that the foregut and midgut of Ae. togoi did not function as a barrier to mf migration from these sites to the thoracic musculature, as observed in normal L1 larvae. Furthermore, all infective L3 larvae obtained from the two experimental feedings were very active and found to distribute in all regions of the head, thorax and abdomen of Ae. togoi.

5. Conclusion This study is the first to clarify the species status of two karyotypic variants of An. kochi collected from five locations in Thailand by using multidisciplinary approaches, including morphological taxonomy, cytogenetics, cross-mating experiments and molecular analyses (rDNA ITS2 and mtDNA COI). These results show that both forms of Thai An. kochi and specimens from other countries (Bhutan, Cambodia and Indonesia) are of the same species. Female An. kochi bear five types of antennal sensilla, identified by SEM. Moreover, the ability of An. kochi to transmit NSP B. malayi was also investigated for the first time, and it was found that this species is a refractory vector. The cibarial armature of An. kochi is an important factor that affects ingested mf, as broken and dead mf were found in the midgut.

Funding This research work was supported by the Anandamahidol Foundation (Year 2016–2019) and Chiang Mai University (CMU) through the Center of Insect Vector Study to A. Saeung, and the Distinguished Research Professor Grant, Thailand Research Fund (grant number DPG6280002) and Khon Kaen University Grant to W. Maleewong (subproject to A. Saeung).

CRediT authorship contribution statement Watchara Jatuwattana: Investigation, Data curation, Writing - original draft preparation. Atiporn Saeung: Conceptualization, Data curation, Formal analysis,

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Writing - original draft preparation, Writing - review and editing, Funding Acquisition. Kritsana Taai, Wichai Srisuka, Kittipat Aupalee, Kanchon Pusawang: Investigation. Petchaboon Poolphol, Sorawat Thongsahuan: Resources. Pradya Somboon: Writing - review and editing. Wanchai Maleewong: Supervision, Funding Acquisition.

Declaration of Competing Interest All authors declare that we have no conflict of interest.

Acknowledgments We would like to thank Dr. Wannapa Suwonkerd, Office of Disease Prevention and Control Region 1st, Ministry of Public Health, Chiang Mai, for providing specimens from Cambodia for this research. We are grateful to Ms. Tippawan Yasanga from the Medical Science Research Equipment Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, for her technical assistance on SEM.

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Author statement

12 December 2019 Dear Professor John Beier Editor Acta Tropica Regarding our manuscript Ref: ACTROP_2019_1433 entitled “Systematic studies of Anopheles (Cellia) kochi (Diptera: Culicidae): morphology, cytogenetics, crossmating experiments, molecular evidence and susceptibility level to infection with nocturnally subperiodic Brugia malayi” for publication as “Research Article” in Acta Tropica.

We trust you will find this paper interesting and appropriate for the journal. It has not been published before and has not been submitted elsewhere.

Your kind consideration of this manuscript would be greatly appreciated.

Yours faithfully,

Atiporn Saeung, Ph.D. Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand Tel: +66-53-935342 E-mail: [email protected]

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behaviour of Anopheles mosquitoes in South Halmahera, Indonesia. Malar. J. 16, 310. Stoops, C.A., Gionar, Y.R., Shinta, Sismadi, P., Elyazar, I.R., Bangs, M.J., Sukowati, S., 2007. Environmental factors associated with spatial and temporal distribution of Anopheles (Diptera: Culicidae) larvae in Sukabumi, West Java, Indonesia. J. Med. Entomol. 44, 543–553. Stoops, C.A., Gionar, Y.R., Shinta, Sismadi, P., Rachmat, A., Elyazar, I.F., Sukowati, S., 2008. Remotely-sensed land use patterns and the presence of Anopheles larvae (Diptera: Culicidae) in Sukabumi, West Java, Indonesia. J. Vector Ecol. 33, 30–39. Suwannamit, S., Baimai, V., Otsuka, Y., Saeung, A., Thongsahuan, S., Tuetun, B., Apiwathnasorn, C., Jariyapan, N., Somboon, P., Takaoka, H., Choochote, W., 2009. Cytogenetic and molecular evidence for an additional new species within the taxon Anopheles barbirostris (Diptera: Culicidae) in Thailand. Parasitol. Res. 104, 905–918. Taai, K., Baimai, V., Thongsahuan, S., Saeung, A., Otsuka, Y., Srisuka, W., Sriwichai, P., Somboon, P., Jariyapan, N., Choochote, W., 2013. Metaphase karyotypes of Anopheles paraliae (Diptera: Culicidae) in Thailand and evidence to support five cytological races. Trop. Biomed. 30, 238–249. Taai, K., Harbach, R.E., 2015. Systematics of the Anopheles barbirostris species complex (Diptera: Culicidae: Anophelinae) in Thailand. Zool J Linnean Soc. 174, 246-264. Taai, K., Harbach, R.E., Aupalee, K., Srisuka, W., Yasanga, T., Otsuka, Y., Saeung, A., 2017. An effective method for the identification and separation of Anopheles minimus, the primary malaria vector in Thailand, and its sister species Anopheles harrisoni, with a comparison of their mating behaviors. Parasit. Vectors. 10, 97. Taai, K., Harbach, R.E., Somboon, P., Sriwichai, P., Aupalee, K., Srisuka, W., Yasanga, T., Phuackchantuck, R., Jatuwattana, W., Pusawang, K., Saeung, A., 2019. A method for distinguishing the important malaria vectors Anopheles dirus and An. cracens (Diptera: Culicidae) based on antennal sensilla of adult females. Trop. Biomed. 34, 1–12.

26

Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Thongsahuan, S., Baimai, V., Otsuka, Y., Saeung, A., Tuetun, B., Jariyapan, N., Suwannamit, S., Somboon, P., Jitpakdi, A., Takaoka, H., Choochote, W., 2009. Karyotypic variation and geographic distribution of Anopheles campestris-like (Diptera: Culicidae) in Thailand. Mem. Inst. Oswaldo Cruz. 104, 558–566. Thongsahuan, S., Otsuka, Y., Baimai, V., Saeung, A., Hempolchom, C., Taai, K., Srisuka, W., Dedkhad, W., Sor-suwan, S., Choochote, W., 2014. Cytogenetic, crossing and molecular evidence of two cytological forms of Anopheles argyropus and three cytological forms of Anopheles pursati (Diptera: Culicidae) in Thailand. Trop. Biomed. 31, 641–653. Van den Broek, I.V., Den Otter, C.J., 1999. Olfactory sensitivities of mosquitoes with different host preferences (Anopheles gambiae s.s., An. arabiensis, An. quadriannulatus, An. m. atroparvus) to synthetic host odours. J. Insect Physiol. 45, 1001–1010. Walton, C., Handley, J.M., Tun-Lin, W., Collins, F.H., Harbach, R.E., Baimai, V., Butlin, R.K., 2000. Population structure and population history of Anopheles dirus mosquitoes in Southeast Asia. Mol. Biol. Evol. 17, 962–974. Wang, G., Li, C., Guo, X., Xing, D., Dong, Y., Wang, Z., Zhang, Y., Liu, M., Zheng, Z., Zhang, H., Zhu, X., Wu, Z., Zhao, T., 2012. Identifying the main mosquito species in China based on DNA barcoding. PLoS One. 7, e47051. World Health Organization., 2007. Anopheline species complexes in South and SouthEast Asia, SEARO Technical Publication No. 57. WHO Regional Office for South-East Asia, New Delhi.

FIGURE LEGENDS

27

Fig. 1 Representation of the metaphase karyotypes of An. kochi. (A) Form A (X1, Y1). (B) a novel Form B (X2, Y2). (C) Diagrammatic representation of Form A (X1, X2, Y1). (D) Diagrammatic representation of novel Form B (X1, X2, Y2).

28

Fig. 2 Maximum likelihood (ML) phylogenetic tree of 24 isolines of An. kochi collected from Thailand (Forms A and B), Bhutan, Cambodia, and Indonesia based on ITS2 sequences, with An. cracens, An. dirus and An. tessellatus as the outgroups. Bootstrap values (ML/NJ) are shown at each node. Branch lengths are proportional to genetic distance (scale bar).

29

30

Fig. 3 Maximum likelihood (ML) phylogenetic tree of 24 isolines of An. kochi collected from Thailand (Forms A and B), Bhutan, Cambodia, and Indonesia based on COI gene sequences, with An. cracens, An. dirus and An. tessellatus as the outgroups. Bootstrap values (ML/NJ) are shown at each node. Branch lengths are proportional to genetic distance (scale bar).

Fig. 4 Scanning electron micrographs of the female antenna of An. kochi. (A) The flagellum consisting of 13 flagellomeres. (B) The scape (Sc), pedicel (Pe), and first flagellomere.

31

Fig. 5 Scanning electron micrograph showing various types of sensilla housed on the female antenna of An. kochi. ac, aculeae; btc, blunt-tipped sensillum trichodeum; lch, large sensillum chaeticum; lco, large sensillum coeloconicum; ltc, long sharp-tipped sensillum trichodeum; sa, sensillum ampullaceum; sb, sensillum basiconicum; sch, small sensillum chaeticum; stc, short sharp-tipped sensillum trichodeum.

32

Fig. 6 Scanning electron micrographs of female antennal sensilla of An. kochi. (A) Sensillum ampullaceum. (B) Sensillum basiconicum. (C) Large sensillum coeloconicum. (D) Small sensillum coeloconicum (sco) at the first flagellomere. (E) Higher magnification of sco at the tip of flagellomere 13.

33

Fig. 7 Representation of B. malayi microfilariae observed in the midgut of infected mosquitoes, dissected at 5 min, 1 h and 12 h after feeding. (A, C) normal mf with intact sheath cuticle and column nuclei of Ae. togoi. (E) normal exsheathed mf of Ae. togoi, dissected at 12 h. (B, D, F) Broken mf with or without nuclei protruding through a ruptured cuticle and sheath of An. kochi, (Black triangle). (G) L1 larvae recovered from thoracic muscle fibres of Ae. togoi, dissected at 96 h. (H) Cibarial armature of An. kochi. c = cibarium; Ct = cibarial teeth; P = pharynx; Pd = pediment.

Table 1

34

Locations, code of isolines, karyotypic forms of An. kochi and their GenBank accession numbers

Location

Code of

Karyotypic

(Geographical coordinate)

isoline

form

Cm-1*

A

Cm-2

Region

GenBank accession number

Reference

ITS2

COI

ITS2, COI

MK881135

MK893403

This study

A

ITS2, COI

MK881136

MK893404

This study

Cm-3

A

ITS2, COI

MK881137

MK893405

This study

Nn-1

A

ITS2, COI

MK881138

MK893406

This study

Nn-2*

A

ITS2, COI

MK881139

MK893407

This study

Nn-3

A

ITS2, COI

MK881140

MK893408

This study

Tr-1

A

ITS2, COI

MK881144

MK893412

This study

Tr-2*

A

ITS2, COI

MK881145

MK893413

This study

Tr-3

A

ITS2, COI

MK881146

MK893414

This study

Pg-1

A

ITS2, COI

MK881141

MK893409

This study

Pg-2

A

ITS2, COI

MK881142

MK893410

This study

Pg-3*

A

ITS2, COI

MK881143

MK893411

This study

Ur-1

B

ITS2, COI

MK881147

MK893415

This study

Ur-2

B

ITS2, COI

MK881148

MK893416

This study

Ur-3*

B

ITS2, COI

MK881149

MK893417

This study

Dw-1

-

ITS2, COI

MK881150

MK893418

This study

Dw-2

-

ITS2, COI

MK881151

MK893419

This study

Dw-3

-

ITS2, COI

MK881152

MK893420

This study

Pc-1

-

ITS2, COI

MK881153

MK893421

This study

Pc-2

-

ITS2, COI

MK881154

MK893422

This study

Pc-3

-

ITS2, COI

MK881155

MK893423

This study

Rt-1

-

ITS2, COI

MK881156

MK893424

This study

An. kochi Thailand Chiang Mai (19°08'59"N 98°54'49"E)

Nan (18°48'39"N 100°53'29"E)

Trang (7°44'47"N 99°23'22"E)

Phang Nga (8°33'22"N 98°27'05"E)

Ubon Ratchathani (14°32'56"N 105°14'34"E)

Dawathang, Bhutan (26°52'05"N 90°31'33"E)

Pucak, Indonesia (5°08'39"S 119°39'21"E)

Ratanakiri, Cambodia

35

(13°51'28"N 107°06'04"E)

Singapore

China

Rt-2

-

ITS2, COI

MK881157

MK893425

This study

Rt-3

-

ITS2, COI

MK881158

MK893426

This study

-

-

COI

-

KF564705

-

-

COI

-

KF564706

-

-

COI

-

KF564707

-

-

ITS2

EU650424

-

unpublished

-

-

COI

-

JQ728290

Wang et al.

Chan et al. (2014) Chan et al. (2014) Chan et al. (2014)

(2012) -

-

COI

-

JQ728292

Wang et al. (2012)

An. cracens

-

-

ITS2

MG008576

-

Wong et al. unpublished

-

-

COI

MG002549

Wong et al. unpublished

An. dirus

-

-

ITS2

KP298432

-

Huong et al. unpublished

-

-

COI

AJ271384

Walton et al. (2000)

An. tessellatus

-

-

ITS2

EU650425

-

Ma and Wu unpublished

-

-

COI

-

KF564699

Chan et al. (2014)

a

Used in cross-mating experiments.

36

Table 2 Cross-mating experiments among the five isolines of An. kochi.

Crosses

Total eggs

Embryonation

No. hatch

No. pupation

No. Emergence

No. females and males from

(Female x Male)

(number)*

rate**

(%)

(%)

(%)

total emergence n (%)

Adult sex

Female

Male

ratio

Parental cross Cm-1A x Cm-1A

120 (37, 83)

97

116 (96.67)

114 (98.28)

114 (100.00)

59 (51.75)

55 (48.25)

1.07

Nn-2A x Nn-2A

121 (59, 62)

98

119 (98.34)

114 (95.80)

112 (98.24)

55 (49.10)

57 (50.90)

0.96

Ur-3B x Ur-3B

111 (62, 49)

99

108 (97.30)

106 (98.15)

106 (100.00)

51 (48.11)

55 (51.89)

0.92

Tr-2A x Tr-2A

133 (45, 88)

94

116 (87.22)

116 (100.00)

115 (99.14)

64 (55.65)

51 (44.35)

1.25

Pg-3A x Pg-3A

107 (44, 63)

100

106 (99.06)

106 (100.00)

100 (94.34)

53 (53.00)

47 (47.00)

1.12

Reciprocal cross Cm-1A x Nn-2A

214 (98, 116)

91

161 (75.23)

150 (93.17)

146 (97.33)

75 (51.37)

71 (48.63)

1.05

Cm-1A x Pg-3A

183 (128, 55)

100

174 (95.08)

151 (86.78)

143 (94.70)

63 (44.06)

80 (55.94)

0.78

Cm-1A x Ur-3B

140 (91, 49)

99

136 (97.12)

132 (97.05)

132 (100.00)

71 (53.79)

61 (46.21)

1.16

Cm-1A x Tr-2A

144 (59, 55)

98

112 (98.25)

106 (94.64)

106 (100.00)

58 (54.72)

48 (45.28)

1.20

Nn-2A x Cm-1A

161 (69, 92)

100

156 (96.89)

145 (92.95)

142 (97.93)

65 (45.77)

77 (54.23)

0.84

Nn-2A x Pg-3A

105 (57, 48)

90

88 (83.81)

79 (89.77)

75 (94.94)

36 (48.00)

39 (52.00)

0.92

Nn-2A x Ur-3B

128 (59, 69)

88

106 (82.81)

99 (93.40)

96 (96.97)

44 (45.83)

52 (54.17)

0.84

Nn-2A x Tr-2A

181 (77, 104)

89

147 (81.21)

146 (99.32)

145 (99.31)

72 (49.66)

73 (50.34)

0.98

Table 2 (continued).

37

Crosses

Total eggs

Embryonation

(Female x Male)

(number)*

rate**

No. hatch (%)

No. pupation

No. Emergence

No. females and males from

(%)

(%)

total emergence n (%)

Adult sex

Female

Male

ratio

Tr-2A x Cm-1A

112 (69, 43)

87

96 (75.81)

94 (95.92)

92 (97.87)

45 (48.91)

47 (51.09)

0.95

Tr-2A x Nn-2A

150 (116, 34)

82

119 (79.33)

113 (94.96)

111 (98.23)

53 (47.55)

58 (52.25)

0.91

Tr-2A x Pg-3A

181 (144, 37)

97

167 (92.27)

167 (100.00)

165 (98.80)

93 (42.08)

128 (57.92)

0.72

Tr-2A x Ur-3B

206 (91, 115)

96

194 (94.17)

190 (97.94)

182 (95.79)

85 (46.70)

97 (53.30)

0.87

Pg-3A x Cm-1A

167 (45, 122)

88

139 (83.23)

135 (97.12)

135 (100.00)

61 (45.19)

74 (54.81)

0.82

Pg-3A x Nn-2A

163 (72, 91)

87

122 (74.85)

116 (95.08)

116 (100.00)

60 (51.72)

56 (48.28)

1.07

Pg-3A x Ur-3B

129 (91, 38)

88

103 (79.84)

103 (100.00)

101 (98.06)

53 (52.48)

48 (47.52)

1.10

Pg-3A x Tr-2A

96 (37, 59)

95

86 (89.58)

84 (97.67)

84 (100.00)

43 (51.19)

41 (48.81)

1.04

Ur-3B x Cm-1A

112 (67, 145)

85

91 (81.25)

91 (100.00)

88 (96.70)

45 (51.14)

43 (48.86)

1.04

Ur-3B x Nn-2A

217 (73, 144)

97

197 (90.78)

185 (93.91)

183 (98.92)

87 (47.54)

96 (52.46)

0.90

Ur-3B x Pg-3A

143 (45, 98)

90

123 (86.01)

123 (100.00)

123 (100.00)

62 (50.41)

61 (49.59)

1.01

Ur-3B x Tr-2A

108 (46, 62)

87

89 (82.41)

88 (98.88)

88 (100.00)

43 (48.86)

45 (51.14)

0.95

Table 2 (continued).

38

Crosses

Total eggs

Embryonation

No. hatch

No. pupation

No. Emergence

No. females and males from

(Female x Male)

(number)*

rate**

(%)

(%)

(%)

total emergence n (%)

Adult sex

Female

Male

ratio

F1- hybrid cross (Cm-1A x Nn-2A)F1 x (Cm-1A x Nn-2A)F1

113 (62, 51)

96

105 (92.92)

102 (97.14)

98 (92.08)

52 (53.06)

46 (46.94)

1.13

(Cm-1A x Pg-3A)F1 x (Cm-1A x Pg-3A)F1

121 (69, 52)

92

109 (90.08)

109 (100.00)

109 (100.00)

56 (51.38)

53 (48.62)

1.05

(Cm-1A x Ur-3B)F1 x (Cm-1A x Ur-3B)F1

145 (36, 109)

87

122 (84.14)

120 (98.36)

119 (99.16)

56 (47.06)

63 (92.54)

0.88

(Cm-1A x Tr-2A)F1 x (Cm-1A x Tr-2A)F1

194 (94, 100)

93

169 (87.11)

167 (98.82)

164 (98.20)

84 (51.22)

80 (48.78)

1.05

(Nn-2A x Cm-1A)F1 x (Nn-2A x Cm-1A)F1

110 (56, 54)

92

99 (90.00)

97 (97.98)

96 (98.97)

48 (50.00)

48 (50.00)

1.00

(Nn-2A x Ur-3B)F1 x (Nn-2A x Ur-3B)F1

206 (62, 144)

96

192 (96.88)

192 (100.00)

190 (98.96)

94 (49.47)

96 (50.53)

0.97

(Nn-2A x Tr-2A)F1 x (Nn-2A x Tr-2A)F1

120 (74, 46)

95

95 (79.17)

94 (98.95)

94 (100.00)

49 (52.12)

45 (47.87)

1.08

(Nn-2A x Pg-3A)F1 x (Nn-2A x Pg-3A)F1

126 (48, 78)

99

91 (91.92)

87 (95.60)

84 (96.55)

41 (48.81)

43 (51.19)

0.95

(Tr-2A x Cm-1A)F1 x (Tr-2A x Cm-1A)F1

188 (144, 44)

92

169 (89.89)

166 (98.22)

166 (100.00)

86 (51.81)

80 (48.19)

1.07

(Tr-2A x Nn-2A)F1 x (Tr-2A x Nn-2A)F1

125 (91, 34)

91

106 (84.80)

106 (100.00)

103 (97.17)

51 (49.51)

52 (50.49)

0.98

(Tr-2A x Pg-3A)F1 x (Tr-2A x Pg-3A)F1

153 (33, 120)

100

147 (96.08)

141 (95.92)

141 (100.00)

69 (48.94)

72 (51.06)

0.95

(Tr-2A x Ur-3B)F1 x (Tr-2A x Ur-3B)F1

132 (77, 55)

86

103 (78.03)

102 (99.03)

98 (96.08)

52 (53.06)

48 (48.94)

1.08

Table 2 (continued).

39

Crosses

Total eggs

Embryonation

No. hatch

No. pupation

No. Emergence

No. females and males from

(Female x Male)

(number)*

rate**

(%)

(%)

(%)

total emergence n (%)

Adult sex

Female

Male

ratio

(Pg-3A x Cm-1A)F1 x (Pg-3A x Cm-1A)F1

203 (134, 69)

89

173 (85.22)

164 (94.80)

161 (98.17)

81 (50.31)

80 (49.69)

1.01

(Pg-3A x Nn-2A)F1 x (Pg-3A x Nn-2A)F1

117 (48, 69)

98

106 (90.60)

104 (98.11)

104 (100.00)

55 (52.88)

49 (47.12)

1.12

(Pg-3A x Ur-3B)F1 x (Pg-3A x Ur-3B)F1

198 (116, 82)

96

186 (93.94)

184 (98.92)

184 (100.00)

88 (47.83)

96 (52.17)

0.91

(Pg-3A x Tr-2A)F1 x (Pg-3A x Tr-2A)F1

169 (109, 60)

87

140 (82.84)

140 (100.00)

139 (99.29)

73 (52.52)

66 (47.48)

1.10

(Ur-3B x Cm-1A)F1 x (Ur-3B x Cm-1A)F1

180 (46, 134)

98

164 (91.11)

164 (100.00)

157 (95.73)

82 (52.23)

75 (47.77)

1.09

(Ur-3B x Nn-2A)F1 x (Ur-3B x Nn-2A)F1

207 (63, 144)

96

195 (94.20)

193 (98.97)

189 (97.93)

91 (48.15)

98 (51.85)

0.92

(Ur-3B x Tr-2A)F1 x (Ur-3B x Tr-2A)F1

107 (94, 13)

91

94 (87.85)

90 (95.74)

89 (98.89)

40 (44.94)

49 (55.06)

0.81

(Ur-3B x Pg-3A)F1 x (Ur-3B x Pg-3A)F1

167 (104, 63)

95

155 (92.81)

150 (96.77)

150 (100.00)

77 (51.33)

73 (48.67)

1.05

* two selective egg-batches of inseminated females from each cross. ** dissection from 100 eggs; n = number.

Table 3 Infective rate and density load of An. kochi 14 days after feeding on blood containing NSP B. malayi microfilariae.

40

Mosquito species

Infective rates

Average No. L3 per

(No.)

infected mosquito

% head

L3-distribution % thorax

% abdomen

(range)

(No.)

(No.)

(No.)

5.50 (1-14)

17.00 (22.08)

28.00 (36.36)

32.00 (41.56)

5.53 (1-40)

70.00 (85.37)

5.00 (6.10)

7.00 (8.54)

Experiment 1 An. togoi

46.67 (14/30)

An. kochi

0 (0/30)

Experiment 2 An. togoi

50.00 (15/30)

An. kochi

0 (0/30)

41