NOTA CIENTÍFICA

New host and distribution for the mosquito parasite Strelkovimermis spiculatus

Nuevo hospedador y distribución del parásito de mosquitos Strelkovimermis spiculatus

Lopez, Rocio P.^*, Leonardo M. Díaz-Nieto^* & Corina M. Berón

Instituto de Investigaciones en Biodiversidad y Biotecnología (INBIOTEC) - CONICET and Fundación para Investigaciones Biológicas Aplicadas (FIBA). Mar del Plata, Argentina. E-mail: cberon@fiba.org.ar
^* These authors contributed equally to this work.

Recibido: 29-X-2015
Aceptado: 14-IV-2016

RESUMEN. Strelkovimermis spiculatus, Poinar & Camino 1986 (Nematoda: Mermithidae) ha sido encontrado parasitando algunos géneros de mosquitos tales como Aedes (Ochlerotatus), Culexy Psorophora. En un proyecto sobre distribución de mosquitos en la Provincia de Buenos Aires, Argentina, fueron encontradas especies de mosquitos parasitadas por nematodos en criaderos naturales, en los alrededores de la ciudad de Mar del Plata. El objetivo de este trabajo es identificar este parásito detectado en esta área de distribución y determinar las especies de mosquitos hospedadoras. Se describe la utilidad de secuencias correspondientes a los genes COI y 18S RNAr-ITS1-5.8S RNAr-ITS2-28S RNAr, en la identificación molecular de este nematodo, como complemento de la identificación de acuerdo con caracteres morfológicos, confirmando la identidad de S. spiculatus. En este trabajo se describe por primera vez a este nematodo infestando larvas de Culexeduardoien un criadero natural de mosquitos, registrando la expansión de la distribución sudeste de este agente de control biológico de poblaciones de mosquitos de importancia sanitaria.

PALABRAS CLAVE: Nematodos; Nuevos registros; Culex eduardoi

ABSTRACT. Strelkovimermis spiculatus, Poinar & Camino 1986 (Nematoda: Mermithidae) was found parasitizing some mosquito genera as Aedes (Ochlerotatus), Culex and Psorophora. In a mosquito distribution project in Buenos Aires, Argentina, we found nematodes infecting mosquito larvae in natural breeding sites in the outskirts of Mar del Plata city. The aim of this work was to identify this parasite in this distribution area and determine the mosquito species host. COI and 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA fragment genes were described and were used for molecular identification of this nematode, confirming the morphological diagnostic traits. In this report, a new host of S. spiculatus, the mosquito larvae of Culex eduardoi was detected, expanding its southeastern distribution.

KEY WORDS: Nematodes; New records; Culex eduardoi

Some nematodes are considered potential agents for biological control programs because they are primarily obligate parasites of arthropods and other invertebrates. In particular, members of the Mermithidae family have proved to be effective in parasitizing natural populations of mosquito larvae (Stock & Goodrich-Blair, 2012). Strelkovimermis spiculatus was found for the first time in Argentina infecting larvae of the floodwater mosquito Aedes (Ochlerotatus) albifasciatus by Poinar & Camino (1986). Thus far, this parasite has been isolated in natural habitats from the immature stages of five species of Culex L., and a few Aedes (Ochlerotatus) sp. and Psorophora sp. collected in La Plata, Buenos Aires Province (34°S, 57-58° W) (García & Camino, 1990; Campos & Sy, 2003; Achinelly & Micieli, 2012; Di Battista et al., 2015). Moreover, it has been demonstrated that this nematode infected some Anopheles sp., Aedes sp. and Culex sp. in laboratory bioassays (García &Camino, 1990; Achinelly & Micieli, 2012).

As part of the mosquito distribution and identification project in General Pueyrredon district, Buenos Aires, Argentina, we found nematodes infecting mosquito larvae in natural breeding sites in the outskirts of Mar del Plata city (37º53`32``S, 57º36`02``W). The aim of this work was to identify this parasite in this new distribution area and determine the mosquito species host.

Mosquito larvae from temporary floodwaters were collected from two natural breeding sites on Provincial Route No 2, in summer 2015. Larvae were separated in individual containers for their identification and to determine the presence and the emergence of nematodes. Larvae and pupae of Aedes albifasciatus, Aedes crinifer (Theobald) and Culex eduardoi Casal & García were analyzed in order to determine parasitism in mosquito immature stages (Table I). The results obtained demonstrated that all the mosquito species were infected. The morphological identification of the nematodes was carried out by the analysis of morphological diagnostic traits through the observation by stereoscopic microscopy (at 100-630 amplification times), confirming the presence of S. spiculatus in all mosquito species (Poinar & Camino, 1986).

Table I. Parasitism rates describing the nematode infection of mosquito larvae species collected in natural breeding sites.

As a complement of the morphological identification, a molecular analysis of the nematodes was carried out. Genomic DNA from individual nematodes from each mosquito species and free parasites belonging to the breeding water were obtained by conventional techniques (Sambrook & Russel, 2001), considering free nematodes, those that were collected in the environment, outside of any host. In order to obtain gene sequences useful for the molecular identification of nematodes, the Polymerase Chain Reaction (PCR) technique was carried out using ribosomal and mitochondrial primers designed for eukaryotes, previously described (Folmer et al., 1994; Díaz-Nieto et al., 2013). Due to the fact that the ribosomal primer combination did not allow the amplification of the nematodes 18S rRNA or 28S rRNA complete genes, a set of specific primers were designed (Fig. 1). Therefore, the alignment of nucleotide sequences of ribosomal genes of different nematodes including mermithids, available in the database of the National Center for Biotechnology Information (NCBI, http:// www.ncbi.nlm.nih.gov) was performed. Consensus regions useful as primer sequences were selected, in some cases with degenerate bases. Primer combinations and PCR conditions are detailed in Table II. The amplification products obtained from the cytochrome c oxidase I (COI) and 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA fragment genes of each mosquito species and free nematodes were sequenced (Macrogen, Korea), manually assembled and deposited in the EMBL database under the accession numbers LN879495 and LN879496, respectively. The results were analyzed by BLASTn and multiple-sequence alignment.

Fig. 1. Nuclear ribosomal genes operon showing the hybridization sites of primers used for the nematodes 18S rRNA - 28S rRNA amplification. The specific primer pairs used in this report for the amplification of the complete sequence are indicated in black arrows.

Table II. Primers used for amplification of fragments corresponding to 18S rRNA, 28S rRNA and COI nematode genes.

The multiple sequences alignments analysis of the 636 bp COI fragment (accession No LN879495) showed 100 % identity with S. spiculatus sequences availably in the GeneBank. On the other hand, the 1550 bp partial sequence obtained with ribosomal primers Ce18SFw38 and Ce28Rew356 (accession No LN879496) (according to Fig. 1), showed 100 % identity only with 276 bp of S. spiculatus sequences (DQ665654, KP270701, KP270702, KP270700, KP270703 and KP270704) present in the Genebank (unpublished sequence and Belaich et al., 2015). However, the complete fragment obtained shows a high identity with 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA partial fragments from other nematodes that were available in sequence databases (like Mermis nigrescens, Pheromermis sp. or Caenorhabditis elegans).

The results obtained from the COI and partial 18S rRNA confirm the molecular identification of S. spiculatus according to the morphological diagnostic traits. Dendograms were built using the Neighbor Joining model of the matrix distance program MEGA5, comparing the obtained sequences with others from public databases (NCBI) (Fig. 2).

Fig. 2. Phylogenetic estimation by Neighbor Joining. A, Using COI and B, 18S rDNA coding-sequences. The phylogenetic trees were constructed with the distance matrix model (Neighbor Joining) of MEGA5 program. The node numbers represent the percentage corresponding to the values of 1000 cycles resampling (bootstrap). Species identified in this report are highlighted in bold text. The accession numbers of the sequences of the databases used for the analysis are listed below the name of each nematode species in brackets.

In this study, we report the infection of C. eduardoi by S. spiculatus for the first time and provide tools to contribute with an accurate molecular identification of nematode mosquito parasites, obtaining the first partial sequence of 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA genes. This detection in the outskirts of Mar del Plata city expanded the southeastern distribution of S. spiculatus.

ACKNOWLEDGEMENTS

We especially thank Prof. Ana Tassi (former professor Universidad Nacional de Mar del Plata) for critical revision of the manuscript and Dr. María Fernanda Achinelly for her help in morphological nematode identification. This work was supported by grants of the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT PICT-2013-0431), Universidad Nacional de Mar del Plata (15/E692-EXA742) and CONICET (PIP 2012 No 112 20110100963).

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