In the isolated islets located in the bay and on the mangrove seafront, the species *Rhagovelia plumbea* is present. It is an exclusively marine species, and it represents one of the nine species of the Salina Group in *Rhagovelia*, as described by Molano *et al*. (2018). *Rheumatobates trinitatis* is also present on the waterfront, but also in areas more protected from mangroves such as in the "Manche à eau" bay, the "Rivière salée" or in the mouths of rivers such as the "Canal des Rotours". It shares the waterfront with *Rhagovelia plumbea*. In general, they never move beyond 20 m from the aerial roots of mangroves tree to avoid being carried away by currents and swells (Cheng & Lewing, 1971). In estuaries with mangroves, three species are present: *Rheumatobates mangrovensis*, *R. imitator* and *Limnogonus franciscanus*. According to our study, *R. mangrovensis* has only been observed at the mouth of the "Grande-Rivière à Goyave". *R. imitator* is one of the so-called «modified» *Rheumatobates* with high sexual dimorphism; males have very arched legs (Hungerford, 1954). The largest groups of *R. imitator* individuals observed were in freshwater ponds in the "Grande-Terre" or in small bodies of water behind mangroves. Some individuals have been found drifting in the "Canal des Rotours" (personal observations).

*Limnogonus franciscanus* is one of the largest species (7 mm in length) and the only macropterous species observed during the study (Figure 2). It has been observed in the ponds of "Grande-Terre" and in the water holes behind the mangroves but also at the mouths of the "Grande-Rivière à Goyave" and the "Canal des Rotours". It is therefore a freshwater species that lives mainly in freshwater lands. But like *Rheumatobates imitator*, it can be carried away by currents and live on thin films of freshwater in river mouths.

This distribution, observed in Guadeloupe, seems to be the same as in Trinidad. The same species are present in the same habitats (Nieser & Alkins-koo, 1991).

In the future, it would be interesting to focus on the physiology and/or metabolism of these insects in order to better understand their biology and their involvement in the mangrove food chain. By studying their physiology, we will also be able to better understand their ability to adapt to these changing environments at the interface between land and sea.

**Acknowledgments:** S. Conjard is funded by a Région Guadeloupe 's grant."

**References:**

Hungerford, H.B., 1954. The genus *Rheumatobates* Bergroth (Hemiptera-Gerridae). Univ. of Kansas Sci. Bull. 36, 529-588.

Cheng, L., Lewin, R.A., 1971. An Interesting Marine Insect, *Rheumatobates*. Pac. Insects 13, 333-341.

Lugo, A.E., Snedaker, S.C., 1974. The Ecology of Mangroves. Annu. Rev. Ecol. Syst. 5, 39-64.

Nieser, N., Alkins-Koo, M., 1991. The water bugs of Trinidad & Tobago. Zoology Dep. Univ. West Indies 1-131.

Molano, F., Morales, I., Moreira, F.F.F., 2018. Two new species and key to the salina group of the genus *Rhagovelia* Mayr, 1865 (Hemiptera: Heteroptera: Veliidae) from Colombia. Zootaxa 4457, 305-314.
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# Mangrove semiaquatic bugs (Hemiptera: Gerroidea) from Guadeloupe in Lesser Antilles: first records and new data on species distribution

Suzanne Conjardª ID, Romain Garrousteᵇ ID, Sylvie D.D. Gustaveª and Olivier Grosª,ᶜ ID

ªInstitut de Systématique, Évolution, Biodiversité (ISYEB), Muséum National d'Histoire Naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, Pointe-à-Pitre, France; ᵇInstitut de Systématique, Évolution, Biodiversité (ISYEB), Muséum National d'Histoire Naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, Paris, France; ᶜC3MAG, UFR des Sciences Exactes et Naturelles, Université des Antilles, Pointe-à-Pitre, France

## ABSTRACT

This study aims to illustrate distribution of semiaquatic bug species in Guadeloupe (Lesser Antilles), as there is still little knowledge about the biodiversity of the island's mangroves. In addition to *Limnogonus franciscanus* Stål, 1859 and *Rheumatobates imitator* Uhler, 1894, four species are newly reported for Guadeloupe: *Brachymetra albinerva* Amyot and Servilles, 1843, *Rhagovelia plumbea* Uhler, 1894, *Rheumatobates mangrovensis* China, 2009 and *R. trinitatis* China, 2009 based on taxonomic and phylogenetic analyses. For all recorded species, we provide detailed data on their ecology and distribution in the region of the investigation.

## ARTICLE HISTORY

Received 26 November 2020
Revised 5 May 2021
Accepted 18 May 2021

## KEYWORDS

Veliidae; Gerridae;
Caribbean; mapping;
ecology; mangrove

## Introduction

Semiaquatic bugs are part of the order Hemiptera of insects. They move on the water surface using their hind legs covered with hair, which increases their buoyancy (Andersen 1982). Semiaquatic bugs have piercing and sucking mouthparts (rostrum) to feed on the bodily fluids of their prey (Heckman 2011). Present on every continent, they can be found in a variety of aquatic environments, such as rivers and lakes (freshwater), in estuaries and mangroves (salt to brackish water), and in oceanic waters (Heckman 2011).

These insects have already been studied in various regions throughout the Caribbean such as Colombia, Florida, Guyana, Mexico, Suriname, and Trinidad (Herring 1961; Drake and Van Doesburg 1966; Nieser 1970; Cheng and Lewin 1971; Andersen and Polhemus 1976; Stoner and Humphris 1985; Molano, Morales, and Moreira 2018). In 2011, Heckman listed the aquatic Hemiptera of South America and defined their distribution in the Caribbean without specifying the islands concerned
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(Heckman 2011). In the Lesser Antilles, only a few studies mention aquatic insects. Hungerford (1954) identified three species of *Rheumatobates* Bergroth, 1892 in Trinidad: *R. imitator* Uhler, 1894, *R. mangrovensis* China, 2009, and *R. trinitatis* China, 2009. The macropterous species *Limnogonus franciscanus* Stål, 1859 was also observed and studied in Trinidad (Nummelin 1997) and during a wildlife survey on the island of St Maarten (Yokoyama 2013). Recently, among the 385 species of Hemiptera reported in Guadeloupe, only three species of the superfamily Gerroidea were mentioned in the last survey published: *Limnogonus franciscanus*, *Microvelia pulchella* Westwood, 1834 and *Rheumatobates imitator* (Meurgey and Ramage 2020). However, the range of these species is not specified, and no marine species are listed. Some genera seem to be specifically adapted to marine environments such as the genus *Rhagovelia* Mayr, 1865 which has been identified throughout the Caribbean (Drake and Van Doesburg 1966; Heckman 2011). Unfortunately, only a few studies have been conducted to date in river mouths and mangroves (from marine fringe to swampy areas).

The marine fringe of mangrove in the Lesser Antilles is made up of a single species of mangrove tree *Rhizophora mangle* Linnaeus, 1753, and tidal variations are low (~30 cm tidal range). These combined two aspects induce a relative stability of the environment (Imbert, Rousteau, and Scherrer 2000; Mantran, Hamparian, and Bouchereau 2009). The mangrove of Guadeloupe is the largest of the Lesser Antilles, about 3000 ha (Imbert et al. 2000) and is mainly located in the Grand-Cul-de-Sac-Marin Bay. This bay is protected by a coral reef and the mangrove is mainly sheltered in the southern part of the bay (Mantran et al. 2009). Despite the coral reef, mangrove islands and the mangrove sea fringe are swept by waves and winds, at least for their windward side. Only a few bodies of water in mangroves and river estuaries are sheltered from these disturbances, such as the Manche à Eau (Mantran et al. 2009) or the mouth of the Canal des Rotours. The Rivière Salée, south of Grand-Cul-de-Sac-Marin Bay, is in fact a seawater canal that cuts the island of Guadeloupe in two (Mantran et al. 2009). Behind the mangrove and located further inland, the swamp forest is mainly composed of *Pterocarpus officinalis* Jacquin, 1763 (Spalding, Blasco, and Field 1997). The swamp forest is waterlogged (3-4‰ of salts) depending on river flooding, rainfall or low tidal range. These different aquatic environments are likely to shelter a great diversity of semiaquatic bugs.

This study aims to identify various semiaquatic bugs present in Guadeloupe and to understand their distribution according to the environment in which they occur.

## Material and methods

The sampling area was centred on the Grand-Cul-de-Sac-Marin Bay. All insect collection sites are located in the maritime area adjacent to the Parc National Guadeloupe, except for the sites of Îlet Fajou and Îlet Christophe located in the heart of the national park. Samples were collected from a boat using a landing net with a 1 mm mesh size according to the two following permits: Parc National Guadeloupe ‘Arrête 2018-35’ and ‘Arrêté 2019-15’. Individuals of each species were collected and placed in collection at the Muséum National d’Histoire Naturelle in Paris (France).
Taxonomic and molecular approaches were used for the identification of the species collected. The taxonomic analysis was performed using various determination keys (Hungerford 1954; Andersen and Polhemus 1976; Heckman 2011; Molano, Mondragón, and Morales 2017; Moreira, Rodrigues, Sites, Cordeiro, and Magalhães 2019). For phylogenetic analysis, DNA extraction was performed on a single individual of each morphotype, COI gene was PCR amplified (Folmer, Black, Hoeh, Lutz, and Vrijenhoek 1994) before sequencing by Eurofins (https://eurofinsgenomics.eu/). Once the species identified, it was possible to map their distribution.

## Results

All individuals collected in the Grand-Cul-de-Sac-Marin Bay, belong to four genera (*Brachymetra* Mayr, 1865, *Limnogonus* Stål, 1868, *Rhagovelia* and *Rheumatobates*) and six species (*Brachymetra albinerva* Amyot and Serville, 1843, *Limnogonus franciscanus*, *Rhagovelia plumbea* Uhler, 1894, *Rheumatobates imitator*, *R. mangrovensis* and *R. trinitatis*). *Brachymetra albinerva*, *Rhagovelia plumbea*, *Rheumatobates mangrovensis* and *R. trinitatis* are recorded for the first time for Guadeloupe while *L. franciscanus* and *R. imitator* have already been reported from the island (Meurgey and Ramage 2020). The semiaquatic bug distribution is presented by species, starting with exclusively marine insects, then the species present in swamp forest and ponds, and finally the transient species present in watercourses.

### *Rhagovelia plumbea* Uhler, 1894

The species was discovered in coastal mangroves and mangrove islets of the Grand-Cul-de-Sac-Marin Bay (Figure 1), mainly in mangroves exposed to waves and wind. It was observed either in small groups of two or three individuals or in colonies of thousands of individuals mixing juveniles and adults, around the roots of *Rhizophora mangle* (supplementary data). Moreover, *Rhagovelia plumbea* can coexist with *Rheumatobates trinitatis*, particularly in the Rivière Salée.

### *Rheumatobates trinitatis* China, 2009

The species was discovered in the Manche à Eau, the Rivière Salée, and the Canal des Rotours watercourses (Figure 1). These marine environments are bordered by mangrove trees composed of *Rhizophora mangle* and are mainly protected against winds and waves.

### *Rheumatobates imitator* Uhler, 1894

This species has been observed evolving in large groups (>100 individuals) on the surface of water bodies in Grande-Terre, particularly at the Maison de la Mangrove. On some occasions, small groups of individuals were observed in the mouth of mangrove waterways such as the Canal des Rotours. This species can coexist with *Limnogonus franciscanus*.
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**Figure 1.** Range mapping of the six species studied in and around Grand-Cul-de-Sac-Marin Bay. The arrows show the small bays where *Rheumatobates trinitatis* China, 2009 were occasionally observed.

**Limnogonus franciscanus Stål, 1859**

The species was discovered in the freshwater holes of swampy forests, in the ponds of the Grande-Terre, but also at the mouths of rivers such as the Grande-Rivière à Goyaves, and the Canal des Rotours. This species can thus coexist with other species depending on its location. *Limnogonus franciscanus* can coexist with *Brachymetra albinerva* in freshwater holes of swamp forests, or with *Rheumatobates imitator* in ponds and pools, or with *R. trinitatis* in river mouths. This pattern of occupation has been observed in the Canal Belleplaine and the Canal des Rotours; *L. franciscanus* and *R. imitator* share the upstream part of the canals while *R. trinitatis* is found at the mouths of these rivers.

**Brachymetra albinerva Amyot and Serville, 1843**

The species was discovered in the water holes existing between the roots of *Pterocarpus officinalis* at the Maison de la Mangrove, in groups of a few individuals. *Brachymetra albinerva* coexists with *Limnogonus franciscanus* when water surfaces between roots decrease during dry periods.

**Rheumatobates mangrovensis China, 2009**

The species was discovered in the mouth of the Grande-Rivière à Goyaves which is characterised by constantly freshwater with shrubby vegetation where they can shelter from the sun. They live in small groups of less than ten individuals.
Discussion

The Hemiptera order includes one of the largest variety of insects in the world, estimated at 89,000 species (Moreira et al. 2019). Aquatic species of the Hemiptera order and the Gerromorpha infraorder have a global distribution. Within the Gerromorpha, it is possible to find marine-only species, saltwater tolerant freshwater species, and freshwater-only species (Schuh and Slater 1995). The genus *Rhagovelia* includes freshwater species, however, nine species are grouped in a 'salina' group (subgenus) which is composed of marine species only (Molano et al. 2018). *Rhagovelia plumbea* is a species perfectly adapted to the marine environment. The presence of a pruinose layer of small silks covering the body protects the insect from the sun's rays and allows it to trap the air when it is submerged by waves (Drake and Van Doesburg 1966; Andersen and Polhemus 1976; Molano et al. 2018). Small fan-shaped silks on its median tarsus ensure stabilisation on the water surface (Drake and Van Doesburg 1966; Andersen and Polhemus 1976). It is therefore not surprising to note the presence of this species in the Grand-Cul-de-Sac-Marin Bay, in poorly protected areas strongly impacted by wave, sea spray, and winds.

Some *Rheumatobates* species are considered marine insects. Of the 34 *Rheumatobates* species described (Hungerford 1954), only six are exclusively marine (Andersen and Polhemus 1976) while the others live in freshwater or euryhaline water (Cheng and Lewin 1971). *Rheumatobates* is undergoing an evolutionary process toward becoming exclusively a marine genus (Andersen and Polhemus 1976). The representatives of *Rheumatobates* do not move more than 10–20 m from the roots of *Rhizophora mangle* in order to avoid displacement by currents and tides (Cheng and Lewin 1971). Hungerford (1954) made the first observations and descriptions of *Rheumatobates* in the Lesser Antilles. He highlighted the presence of *R. trinitatis* on the island of Trinidad, in coastal mangroves, as well as the presence of *R. mangrovensis* in the mouth of the great Yarra River (Hungerford 1954). *Rheumatobates imitator* was also observed in Trinidad in the Hollis Reservoir, an inland freshwater pond (Hungerford 1954). These observations in Trinidad are similar to the distribution currently observed in Guadeloupe. Thus, *R. trinitatis* lives in saltwater and coastal mangroves, *R. mangrovensis* in the mouths of large rivers, and *R. imitator* in freshwater pools behind mangroves and deeper into the lands of Grande-Terre.

*Limnogonus franciscanus* has only been observed in small populations (<10 individuals) in the Canal des Rotours and at the mouth of the Grande-Rivière à Goyaves. On the other hand, this species is easily observed in freshwater areas behind the mangrove swamps and in freshwater ponds through Grande-Terre. Its presence in river mouths is potentially linked to a transfer between the freshwater pools of Grande-Terre, the water holes behind the mangroves and the river mouths. The light topography, the rains – which can be abundant – and low tides (maximum 30 cm tidal range) can allow individuals to drift on a thin film of freshwater to the river mouths in contact with marine environments. *Limnogonus franciscanus* has also been observed in Trinidad and St Maarten (Nummelin 1997; Yokoyama 2013) but this drifting phenomenon has not yet been described.

The reason for the distribution of these insects may be related to various factors such as (i) the water salinity, (ii) the swell, (iii) their adaptation to warmth and (iv) their predation capacities and food choices. Ditrich and Papáček (2016) observed and
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compared the predation behaviour of gerrids and veliids in temperate environments. Veliids, which live in rivers, are sharper and faster in their predation behaviour to adapt to the turbulent environment of the rivers. Unlike the veliids, gerrids live in a lentic environment and do not need to be fast in their predation (Ditrich and Papáček 2016). In Guadeloupe, the veliid *Rhagovelia plumbea* lives in open water disturbed by waves. They are adapted to life in an environment not yet occupied by other subaquatic bugs where preys are available. Conversely, the gerrids of *Rheumatobates* spp. live in less turbulent waters found in mangrove bays. The marine species of Gerroidea can be separated into two groups, coastal and pelagic species (Cheng 2006) and their trophic regime is not well known. Coastal species feed on small insects from the land: such as collembolas, copepods, midges or any insects that fall to the water surface (Andersen and Polhemus 1976). Pelagic species of the *Halobates* Eschscholtz, 1822 genus feed on zooplankton trapped at the surface of the sea but also on small fishes, anemones or jellyfishes (Andersen and Cheng 2004). These semi-aquatic bugs (coastal and/or pelagic species) are opportunistic and adaptable, they feed on what they find and it is hard to determine their diet. Molecular-based approach, such as metabarcoding, is used to assess the diet composition of some arthropods and for understanding the trophic network of the environments they occupy (Sow, Haran, Benoit, Galan, and Brévault 2020). Like other molecular based approaches, metabarcoding only gives qualitative results on the presence/absence of prey species in the gut or faecal samples of various animals (Greenstone, Payton, Weber, and Simmons 2014) but this could represent a very nice opportunity to determine in a near future the diets of the semiaquatic bugs studied here.

This study allows us to identify the semiaquatic bugs species found in the mangrove environment (marine fringe and swamps in further inland) of Guadeloupe and it also allows us to determine their preferred environments and the interactions that can occur between them. In the future, it would be interesting to focus on the species' physiology and metabolism in order to improve our knowledge and better understand their implications on Guadeloupe's mangroves and by their capacity to give indications on the state of the fragile environment (disturbances, pollution, climate change) that mangroves constitute. By studying their physiology, we will thus be able to better understand their capacity to adapt to these potentially changing environments at the interface between land and sea.

**Disclosure statement**

No potential conflict of interest was reported by the author(s).

**Funding**

S. Conjard was supported by a grant from ‘Conseil Régional de la Guadeloupe’.

**ORCID**

Suzanne Conjard http://orcid.org/0000-0001-8344-7024
Romain Garrouste http://orcid.org/0000-0002-0880-5781
Olivier Gros http://orcid.org/0000-0002-2297-2009

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RESEARCH ARTICLE

# Evidence of new strains of *Wolbachia* symbiont colonising semiaquatic bugs (Hemiptera: Gerroidea) in mangrove environment of the Lesser Antilles

Suzanne Conjard<sup>1*</sup>, Damien F. Meyer<sup>2,3*</sup>, Rosalie Aprelon<sup>2,3</sup>, Nonito Pagès<sup>2,3</sup>, Olivier Gros<sup>1</sup>

1 Institut de Systématique, Évolution, Biodiversité (ISYEB), Muséum national d'Histoire naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles Pointe-à-Pitre, Guadeloupe, France, 2 CIRAD, UMR ASTRE, Petit-Bourg, Guadeloupe, France, 3 ASTRE, Université Montpellier, CIRAD, INRA, Montpellier, France

* suzanne.conjard@gmail.com (SC); damien.meyer@cirad.fr (DFM)

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Check for
updates

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OPEN ACCESS

**Citation:** Conjard S, Meyer DF, Aprelon R, Pagès N, Gros O (2022) Evidence of new strains of *Wolbachia* symbiont colonising semiaquatic bugs (Hemiptera: Gerroidea) in mangrove environment of the Lesser Antilles. PLoS ONE 17(8): e0273668.
https://doi.org/10.1371/journal.pone.0273668

**Editor:** Olle Terenius, Uppsala University; Uppsala Universitet, SWEDED

**Received:** November 9, 2021

**Accepted:** August 12, 2022

**Published:** August 30, 2022

**Copyright:** © 2022 Conjard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** The data underlying the results presented in the study are available from https://www.ncbi.nlm.nih.gov/nuccore/MW019457 and https://www.ncbi.nlm.nih.gov/nuccore/MW114524.

**Funding:** This research was funded in the frame of the projects MALIN, grant number 2018-FED-1084, and INSULA, grant number 2020-FED-45, both funded by the European Union and the Guadeloupe Regional Council under the European Research and Development Funds (ERDF) 2014-

## Abstract

*Wolbachia* Hertig, 1936 is an intracellular bacterial symbiont colonizing many arthropods. Of the studies done on the bacteria present in the superfamily Gerroidea Leach, 1815, no report of *Wolbachia* infection had yet been made. Thus, we checked the presence of *Wolbachia* in six Gerroidea species which colonize tropical aquatic environments by PCR using *wsp* primer set before sequencing and phylogenetic analyses. Insects were collected in the marine fringe of mangroves, in river estuaries, in swampy mangroves, and in ponds from Guadeloupe islands (Caribbean). Two new strains of *Wolbachia* were detected in these Gerroidea. They were named *wLfran* and *wRmang*. The *wsp* sequences suggest that the strains belong to the already described E supergroup or similar. *wLfran* is present in *Limno-gonus franciscanus* Stål, 1859 and *Rheumatobates trinitatis* (China, 1943) while *wRmang* appears to be present exclusively in *R. mangrovensis* (China, 1943). Three other species were analysed, but did not appear to be infected: *Brachymetra albinerva* (Amyot & Servile, 1843), *Halobates micans* Eschscheltz, 1822, and *Microvelia pulchella* Westwood, 1834. The results presented here highlight for the first time the presence of new intracellular *Wolbachia* strains in Gerroidea colonising tropical aquatic environments like mangrove habitats from inlands to sea shore.

## Introduction

*Wolbachia pipientis* commonly known as *Wolbachia*, is an alpha-proteobacterium belonging to the Rickettsiales Gieszczykiewicz, 1939 order. It is mainly present intracellularly in the geni-tal and somatic tissues of more than 60% of arthropods [1] and in nematodes [2]. In arthro-pods, the *Wolbachia* bacterium is mainly known for its ability to manipulate reproduction of the infected host, and can lead to production of infected females without males (inducing
2020 program. https://ec.europa.eu/info/research-and-innovation/funding/funding-opportunities/funding-programmes-and-open-calls_en#erdf
Suzanne Conjard was supported by a grant from the Guadeloupe Regional Council. https://www.regionguadeloupe.fr/accueil/#_ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing interests:** The authors have declared that no competing interests exist.

parthenogenesis), feminization of genetic males (inducing feminization), destruction of infected male offspring (killing males) and cytoplasmic incompatibility between infected males and uninfected (or infected with another strain) females [3].

To detect the presence of *Wolbachia*, a couple of methods are possible. The FISH (Fluorescence *In Situ* Hybridization) technique with the use of specific probes to visualize the bacteria within the insect tissues [4,5], or PCR (Polymerase Chain Reaction) targeting specific genes. The first detections of *Wolbachia* thanks to PCR were made using universal 16S rDNA primer sets and then using the *ftsZ* gene, which was considered more specific at the time [1,6]. Subsequently, PCR targeting the specific *wsp* gene (*Wolbachia* surface protein) which encodes a major protein of the cell surface layer became widespread [7]. Primers encoding *wsp* allow a preliminary identification of the *Wolbachia* strain. However, these last two decades the MLST (Multilocus Sequence Typing) method, which combines a set of genes, has become more popular [8]. This technique avoids the high recombination rates found with the *wsp* technique, which allows for a more reliable phylogeny [9]. More recently though the MLST and *wsp* typing system has been highly criticized, and whole genome sequencing and phylogenomic approaches seem to be best [10,11].

The phylogeny of *Wolbachia* is divided in various supergroups. Early phylogenetic analyses done almost 30 years ago determined the presence of two supergroups A and B, grouping together *Wolbachia* strains infecting different arthropods [1]. Then, *Wolbachia* strains phylogenetically related to supergroups C and D have been observed in filarial nematode hosts [12]. Today, the use of the MLST technique allows the identification of 19 supergroups up to the letter S [13].

Insects of the super-family Gerroidea are semi aquatic bugs belonging to the order Hemiptera. They live on the surface of the water, in rivers, estuaries or even in the sea. In tropical environments, they are also present in mangroves [14]. These semiaquatic insects move on the surface of the water thanks to a bunch of small bristles at the end of their legs [15]. They are sucking biting insects that feed opportunistically depending on the prey present on the surface of the water [15,16].

Apart from the wealth of publication on *Wolbachia* in many fields, there are only few studies on aquatic Hemiptera. According to research in Missouri, 52% of aquatic insects are infected with *Wolbachia*, compared to 60% in terrestrial insects [17]. Few recent studies demonstrated the presence of *Wolbachia* in semiaquatic bugs. In Cameroon, Esemu *et al.* (2019) detected *Wolbachia* in freshwater insects that represent a potential reservoir for the bacterium *Mycobacterium ulcerans* MacCallum, 1948 causing Buruli ulcer. This research focused on nine species of the Gerridae Leach, 1815 and Veliidae Amyot & Serville, 1843 families including the three genera *Limnogonus*, *Microvelia*, and *Rhagovelia* [18]. More recently, Castillo *et al.* [19] described the microbiome (including *Wolbachia*) associated with six species of gerrids belonging to the genera *Platygerris*, *Potamobates*, *Rheumatobates*, and *Telmatometra* from Panama [19].

Semi-aquatic bugs in the Lesser Antilles are not well known. A few studies confirmed their presence in these islands, the first focusing on Trinidad [20] and the second on Saint Martin [21]. For Guadeloupe, only two recent studies listing the insects of the island confirm the presence of six Gerroidea: *Brachymetra albinerva*, *Limnogonus franciscanus*, *Microvelia pulchella*, *Rhagovelia plumbea*, *Rheumatobates mangrovensis* and *R. trinitatis* [14,22]. Our study on *Wolbachia* in the Gerroidea of Guadeloupe focuses on six gerrids. *Brachymetra albinerva* lives in puddles between the roots of *Pterocarpus officinalis* in the swamp forest. *Limnogonus franciscanus* has the largest range, occurring on the terrestrial fringe of the mangrove, in the swamp forest and in the freshwater pools of “Grande-Terre” [14]. *Halobates micans* is exclusively marine, occurring in waves on the coast [23]. *Microvelia pulchella* is present in freshwater pools [22]. *Rheumatobates mangrovensis* is only present downstream of the “Grande-Rivière à Goyaves”.
*Rheumatobates trinitatis* is also a marine species but prefers the mangrove waters more pro-
tected from wind and swell by mangrove trees, *Rhizophora mangle* Linnaeus, 1753 [14].

There is a great variability of *Wolbachia* strains in Arthropods [24]. Depending on geo-
graphical isolation or living environment, a host or strain of *Wolbachia* will change. For exam-
ple, the fly *Bactrocera dorsalis* Hendel, 1912 in China is infected by four different strains
throughout the country [25] or three species of the same fly genus can share one strain in
South Asia [26]. Knowing this, it is interesting to demonstrate the presence of the bacterium in
Gerroidea of Guadeloupe in order to have a first idea of the prevalence of the bacterium on the
island. In order to detect the presence of *Wolbachia* symbionts in Gerroidea, we solely use
PCR with primers specific for the *wsp* gene.

**Materials and methods**

Sampling

Sampling took place between 2019 and 2021, in the mangrove of the “Grand-Cul-de-Sac-Marin”, in the swampy forest of the back mangrove at the “Maison de la Mangrove”, in a pond of Grande-Terre “Étang Fréchou”, and on the shoreline coast of the Atlantic side of Grande-Terre “Anse-Maurice” (Fig 1). The samples were taken by boat in the marine mangrove and by feet for the other sites, using a 1 mm-mesh size nylon net. Apart from the collection sites “Anse-Maurice” and “Étang Fréchou”, all other insect collection sites are located in the marine area adjacent to the “Parc National de la Guadeloupe” or in the case of the “Grande-Rivière à Goyaves” estuary in the heart of the “Parc National de la Guadeloupe” (“permit n°2019-15”).

Map of wetlands in the
Grand-Cul-de-Sac-Marin
and sampling locations

<figure><img src="image_136.png" /><figcaption>**Fig 1. Map of wetlands in Guadeloupe and associated sampling sites.** https://carto.karugeo.fr/1/layers/zones_humides_971.map.

https://doi.org/10.1371/journal.pone.0273668.g001</figcaption></figure>
A total of 60 individuals of Gerroidea were collected and analyzed in different sampling sites according to their habitats (Fig 1 and Table 1). Where possible, for each collection, males, females and juveniles were analyzed.

## Identification of insects, PCR amplification, and sequencing

The collected Gerroidea were individually identified using integrative taxonomy with two methods. Taxonomic determination keys [15,23,27–30] and PCR amplification followed by sequencing of the PCR products of the gene encoding COI (Cytochrome Oxidase subunit I) using the primers set LCO1490F and HCO2198R [31]. DNA was extracted using the DNeasy Blood & Tissue Kit following the manufacturer's protocol. PCR was performed under the following conditions: initial denaturation at 94°C for 4 minutes, 30 denaturation cycles (94°C, 60 seconds), annealing (55°C, 45 seconds), extension (72°C, 45 seconds) and a final extension at 72°C for 1 minute. The expected size of the PCR products was checked after 2% agarose gel electrophoresis before to be directly sequenced by Eurofins (http://www.eurofinsgenomics.eu).

When identified, insects were selected in a representative manner according to species and habitat location. The primer set used for PCR was that corresponding to the *Wolbachia wsp* -specific surface protein (wsp81F and wsp691R) developed by Duron et al. (2008) [32]. PCR was performed under the following conditions: initial denaturation at 94°C for 3 minutes, 40 denaturation cycles (94°C, 60 seconds), annealing (54°C, 60 seconds), extension (72°C, 1 minute) and a final extension at 72°C for 7 minutes. The expected size of the PCR product is 610 bp. PCR products were verified after electrophoresis on 2% agarose gel electrophoresis. The positive control used is DNA from *Rheumatobates mangrovensis* that has been shown to be positive. The negative control is ultra-pure water. The PCR products were then directly sequenced (from the both primers used) by Eurofins (http://www.eurofinsgenomics.eu).

## Phylogenetic analysis

*Wolbachia wsp* sequences from gerrids used for phylogenetic analysis were downloaded from NCBI (Table 3 and Fig 2) in addition to the sequences obtained in this study (MW019457 and MW114524). They were all analyzed and aligned with Geneious (Geneious version R10, https://www.geneious.com) and MAFFT [33]. The phylogenetic tree was constructed using PhyML software [34] with a maximum likelihood approach. The bootstrap analysis was performed with 1000 replicates, and the bootstrap values were calculated using a 50% majority rule.

<table><caption>Table 1. Number of samples per species and per sampling site.</caption><thead><tr><th>Species</th><th>Sampling site (Fig 1)</th><th>Number of samplings</th></tr></thead><tbody><tr><td><em>Brachymetra albinerva</em></td><td>Maison de la Mangrove</td><td>6</td></tr><tr><td><em>Halobates micans</em></td><td>Anse-Maurice</td><td>6</td></tr><tr><td rowspan="3"><em>Linnogonus franciscanus</em></td><td>Canal des Rotours</td><td>10</td></tr><tr><td>Rousseau</td><td>2</td></tr><tr><td>Maison de la Mangrove</td><td>8</td></tr><tr><td><em>Microvelia pulchella</em></td><td>Étang Fréchou</td><td>5</td></tr><tr><td><em>Rheumatobates mangrovensis</em></td><td>Estuary of "Grande-Rivière à Goyaves"</td><td>8</td></tr><tr><td rowspan="5"><em>R. trinitatis</em></td><td>Baie à Chat</td><td>3</td></tr><tr><td>Canal des Rotours</td><td>2</td></tr><tr><td>Ilet Macou</td><td>1</td></tr><tr><td>Manche à Eau</td><td>8</td></tr><tr><td>Rivière Salée</td><td>1</td></tr></tbody></table>

https://doi.org/10.1371/journal.pone.0273668.t001
Tree scale: 0.1

<figure><img src="image_137.png" /><figcaption>Fig 2. Phylogenetic tree of *Wolbachia* detected in Gerroidea and other insects based on their *wsp* sequences. Tree was inferred from maximum likelihood method using PhyML program and drawn using iTOL program. Branches are labelled as Supergroup Letter and specie names followed by GenBank accession numbers. Bootstrap values (1,000 replicates) are indicated, except for branches with a value less than 0.5.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0273668.g002

# Results

## *Wolbachia* infection in Gerroidea of Guadeloupe

*Wolbachia* were detected in three of the six species tested: *L. franciscanus*, *R. mangrovensis* and *R. trinitatis*. *Wolbachia* was detected in all *R. mangrovensis* and *L. franciscanus* individuals while occasionally in *R. trinitatis* and not detected in *B. albinerva*, *H. micans* and *M. pulchella* tested individuals (Tables 2 and 3).

## Sequence and phylogenetic analyses

We amplified and partially sequenced the *wsp* gene (Table 3). From these sequences, two major *Wolbachia* strains were detected in Gerroidea from Guadeloupe and registered in the

<table><caption>Table 2. Table showing the prevalence of *Wolbachia* in Gerroidea samples collected at different collection sites.</caption><thead><tr><td>Species</td><td>Number of <i>Wolbachia</i> negative individuals</td><td>Number of <i>Wolbachia</i> positive individuals</td><td>Sequenced samples</td></tr></thead><tbody><tr><td><i>Brachymetra albinerva</i></td><td>6</td><td>0</td><td>0</td></tr><tr><td><i>Halobates micans</i></td><td>6</td><td>0</td><td>0</td></tr><tr><td><i>Limnogonus franciscanus</i></td><td>0</td><td>14</td><td>6</td></tr><tr><td><i>Microvelia pulchella</i></td><td>6</td><td>0</td><td>0</td></tr><tr><td><i>Rheumatobates mangrovensis</i></td><td>0</td><td>8</td><td>5</td></tr><tr><td><i>R. trinitatis</i></td><td>6</td><td>9</td><td>3</td></tr></tbody></table>

https://doi.org/10.1371/journal.pone.0273668.t002
<table><caption>Table 3. Survey of the sequences obtained and their existing matches in BLAST.</caption><thead><tr><th colspan="4">Results of the study</th><th colspan="3">BLAST match</th></tr><tr><th>Species</th><th>Sample number</th><th>Sampling site (Fig 1)</th><th>Number of nucleotides obtained</th><th>Species name</th><th>Percent identity</th><th>Accession number</th></tr></thead><tbody><tr><td>Limnogonus franciscanus</td><td>#1</td><td>Canal des Rotours</td><td>467</td><td>Dichocrocis punctiferalis</td><td>96.78%</td><td>GU166597.1</td></tr><tr><td>Limnogonus franciscanus</td><td>#2</td><td>Maison de la Mangrove</td><td>434</td><td>Pteromalus puparum</td><td>97.51%</td><td>DQ493917.1</td></tr><tr><td>Limnogonus franciscanus</td><td>#3</td><td>Maison de la Mangrove</td><td>439</td><td>Dichocrocis punctiferalis</td><td>97.27%</td><td>GU166597.1</td></tr><tr><td>Limnogonus franciscanus</td><td>#4</td><td>Maison de la Mangrove</td><td>490</td><td>Pteromalus puparum</td><td>97.53%</td><td>DQ493917.1</td></tr><tr><td>Limnogonus franciscanus</td><td>#5</td><td>Maison de la Mangrove</td><td>512</td><td>Pteromalus puparum</td><td>97.45%</td><td>DQ493917.1</td></tr><tr><td rowspan="3">Limnogonus franciscanus</td><td rowspan="3">#6</td><td rowspan="3">Rousseau</td><td rowspan="3">517</td><td>Ceroptres cerri</td><td>97.25%</td><td>AY095157.1</td></tr><tr><td>Dichocrocis punctiferalis</td><td>96.63%</td><td>GU166597.1</td></tr><tr><td>Dichocrocis punctiferalis</td><td>96.90%</td><td>GU166597.1</td></tr><tr><td rowspan="3">Rheumatobates trinitatis</td><td rowspan="3">#1</td><td rowspan="3">Canal des Rotours</td><td rowspan="3">489</td><td>Pteromalus puparum</td><td>97.73%</td><td>DQ493917.1</td></tr><tr><td>Ceroptres cerri</td><td>97.53%</td><td>AY095157.1</td></tr><tr><td>Dichocrocis punctiferalis</td><td>96.93%</td><td>GU166597.1</td></tr><tr><td rowspan="3">Rheumatobates trinitatis</td><td rowspan="3">#2</td><td rowspan="3">Îlet Macou</td><td rowspan="3">508</td><td>Pteromalus puparum</td><td>97.43%</td><td>DQ493917.1</td></tr><tr><td>Ceroptres cerri</td><td>97.23%</td><td>AY095157.1</td></tr><tr><td>Dichocrocis punctiferalis</td><td>96.65%</td><td>GU166597.1</td></tr><tr><td rowspan="3">Rheumatobates trinitatis</td><td rowspan="3">#3</td><td rowspan="3">Manche à Eau</td><td rowspan="3">512</td><td>Pteromalus puparum</td><td>97.25%</td><td>DQ493917.1</td></tr><tr><td>Ceroptres cerri</td><td>97.05%</td><td>AY095157.1</td></tr><tr><td>Dichocrocis punctiferalis</td><td>96.48%</td><td>GU166597.1</td></tr><tr><td>Rheumatobates mangrovensis</td><td>#1</td><td>Estuary of "Grande-Rivière à Goyaves"</td><td>358</td><td>Delphacodes kuscheli</td><td>93.58%</td><td>KM386825.1</td></tr><tr><td>Rheumatobates mangrovensis</td><td>#2</td><td>Estuary of "Grande-Rivière à Goyaves"</td><td>518</td><td>Macrolophus pygmaeus</td><td>94.56%</td><td>FJ374283.1</td></tr><tr><td>Rheumatobates mangrovensis</td><td>#3</td><td>Estuary of "Grande-Rivière à Goyaves"</td><td>518</td><td>Stephanitis pyrioides</td><td>94.56%</td><td>AB109622.1</td></tr><tr><td>Rheumatobates mangrovensis</td><td>#4</td><td>Estuary of "Grande-Rivière à Goyaves"</td><td>571</td><td>Delphacodes kuscheli</td><td>93.33%</td><td>KM386825.1</td></tr><tr><td>Rheumatobates mangrovensis</td><td>#5</td><td>Estuary of "Grande-Rivière à Goyaves"</td><td>595</td><td>Macrolophus pygmaeus</td><td>93.21%</td><td>FJ374283.1</td></tr></tbody></table>

https://doi.org/10.1371/journal.pone.0273668.t003

NCBI database: wRmang_isolateGuadeloupe (MW019457) and wLfran_isolateGuadeloupe (MW114524).

The wRmang strain was detected exclusively in *Rheumatobates mangrovensis* species, from the unique place where they can be found at the mouth of the river "Grande-Rivière à Goyaves" (Fig 1 and Table 1). The wLfran strain is the most prevalent strain and is detected in *Limnogonus franciscanus* and *R. trinitatis*. The two species do not share the same habitat. However, they can be found occasionally at sites such as the "Canal des Rotours" (Fig 1), a water-course that crosses many environments, wet meadows, swamp forest and leads to mangroves. *Limnogonus franciscanus* is a freshwater species that prefers habitats like puddles or even private fountains in gardens like small or a natural pond at the "Maison de la Mangrove". *Rheumatobates trinitatis* is a marine species that will prefer sheltered mangrove environments such as at the "Manche à Eau" or in the "Baie à Chat" (Fig 1). The two strains have 93% homogeneity between them.
The phylogenetic tree, based on *wsp* gene sequences, shows the distinct presence of *wLfran* and *wRmang* strains within the cluster of E-supergroup sequences (Fig 2).

Discussion

In the present study, six species of semi-aquatic bugs were analyzed by PCR and sequenced to detect and identify *Wolbachia* among Gerroidea from Guadeloupe island (Caribbean, Lesser Antilles). Thus, three Gerroidea species are infected by two new strains (*wLfran* and *wRmang*) belonging to E supergroup. This study provides first insight for the prevalence of *Wolbachia* in Gerroidea in Guadeloupe.

*Wolbachia* strains have been reported on arthropod pests such as scorpion [35], flies [36], terrestrial isopods [4], and spiders [37]. However, the majority of these studies have focused on insects with an impact on health or agriculture. *Wolbachia* has been detected in a wide diversity of mosquito species, some of them vectors of medically important arboviruses [38]. The *Wolbachia* bacterium has been proposed as a regulatory tool for Zika and/or dengue bearing mosquitoes [39–41]. Studies on the occurrence of *Wolbachia* in insect pests of crops mainly concern Diptera [25,26], Acari [24,42] and phytophagous Hemiptera [43–48]. Of all the studies carried out, very few have been conducted on aquatic insects and even fewer on marine insects. However, two recent studies conducted in Cameroon [18] and Panama [19] looking for the bacterial community in the Gerridae and Veliidae found *Wolbachia* in these taxa.

In Guadeloupe, *Wolbachia* has already been studied in some mosquito species: *Aedes aegypti* Linnaeus, 1762, *A. taeniorhynchus* (Wiedemann, 1821) and *Culex quinquefasciatus* Say, 1823 mosquitoes [49,50], *Deinocerites* sp. Theobald, 1901, *Mansonia* sp. Blanchard, 1901, *Ochlerotatus* sp. Reinert, 2000 and *Uranotaenia* sp. Lynch Arribálzaga, 1891 [51]. While mosquitoes of the genus *Aedes* are usually free of *Wolbachia*, this is not the case for *Cx. quinquefasciatus*, which has a variable infection rate of between 79 to 96% [49]. Mosquitoes, although temporarily aquatic insects, do not appear to share common *Wolbachia* strains with Gerroidea based on results of the present study.

The low number of Gerroidea samples collected during this study does not allow statistical treatment of data. However, these preliminary results suggest a high presence of *Wolbachia* in certain species such as *L. franciscanus*, *R. mangrovensis* and *R. trinitatis* and an absence, or very low presence, in *B. albinerva*, *H. micans* and *M. pulchella*. Given that the sampling effort is really different between Gerroidea and Guadeloupean mosquitoes, a pattern of infection appears to be emerging. Like the *Aedes* mosquitoes from Guadeloupe, *B. albinerva* and *M. pulchella*, while sharing the same environment as the other Gerroidea tested, does not appear to be infected. Thus, the presence of *Wolbachia* seems to be related to the insect species rather than to the environment occupied. *Wolbachia* infection is known to be variable within a population of the same genus but also within a same species depending on its geography [25,26,44]. Depending on its geographical origin, the frequency of *Wolbachia* infection can be very variable, ranging from 4–100% in one sampling area to 37–100% in another [44]. Statistically, the intraspecific infection rate can be very high or very low and there is no average infection rate [52]. In some genera, depending on the species, the average infection rate varies from quite low at less than 3% [24] to very high with an infection rate close to 100% [47]. In view of the different forms of infection, it is therefore normal to observe variations in infection among the six species studied in Guadeloupe.

Currently, taking into account all invertebrates and all analytical methods, there are nineteen *Wolbachia* supergroups named from A to S (G is not included because it is a grouping of
A and B supergroups) [13,24,26,42]. In our study, *Wolbachia* symbionts infecting mangrove
Gerroidea belong to E supergroup according to *wsp* phylogenetic analysis (Fig 2).

The *Wolbachia* strain colonizing the pirate bug *Orius minutus* (Linnaeus, 1758) is also part of E supergroup. This Anthochorid belongs to the order Hemiptera, but remains genetically distant from the ones analyzed in this study as is not part of the superfamily Gerroidea. In contrast, *Gerris* sp. Fabricius, 1794 from temperate habitats has been found to be positive for *Wolbachia* strains of the B supergroup in England [53] but negative in France [32]. Overall, few studies have been conducted and updated on *Wolbachia* E supergroup, but it seems that several hosts are possible, including insects, spiders [37] and springtails [54]. Therefore, there is currently little data regarding the occurrence of *Wolbachia* in the superfamily Gerroidea and the prevalence of E supergroup is not yet known. Typing of strains into supergroups was mainly based on phylogenetic inference of the *Wolbachia* surface protein (*wsp*) which is easy to implement and reproducible. However, the small size of this gene and its high recombination rate make it unreliable for precise phylogenetic analyses [8]. Therefore, a full characterization of the MultiLocus Sequence Typing (MLST) is necessary for a correct typing of the strain [8]. MLST analysis is based on the study of several genes. This technique allows refining the analysis by increasing the number of supergroups detected, 16 different supergroups have been described so far, without changing the overall phylogeny [26,55]. However, this technique is nowadays discussed because the markers used are not discriminating enough and are not reliable enough at fine-scale [10].

It is now possible to state that Gerroidea from Guadeloupe mangroves are infected by dif-
ferent *Wolbachia* strains. However, as the use of *wsp* gene sequences is not sufficiently discrim-
inating, it would be interesting to strengthen the research with other methods such as the
analysis of the whole genome sequence of the strains which remains the most reliable method
[10,56]. This later technique would allow us to consolidate the results and possibly confirm the
presence of these strains in E supergroup.

Knowledge of aquatic insects in marine, brackish and lacustrine environments in the tropics is poor. In Guadeloupe, other *Wolbachia* strains have been observed in spiders and ants from swamp and mangrove forests [57]. Increasing our knowledge on the diversity of *Wolbachia* strains colonizing various insects from similar environments through the island would allow us to demonstrate the strategy used by these symbiotic *Wolbachia* strains in such tropical Hemiptera to be transmitted to the new host generations.

Nucleotide sequence accession number

*Wolbachia wsp* gene partial sequences obtained in this study were deposited in the Genebank database under accession numbers MW019457 and MW114524.

Author Contributions

Conceptualization: Suzanne Conjard, Damien F. Meyer, Nonito Pagès, Olivier Gros.

Data curation: Suzanne Conjard, Damien F. Meyer.

Formal analysis: Suzanne Conjard, Damien F. Meyer, Nonito Pagès, Olivier Gros.

Funding acquisition: Nonito Pagès, Olivier Gros.

Investigation: Suzanne Conjard, Olivier Gros.

**Methodology:** Suzanne Conjard, Damien F. Meyer, Rosalie Aprelon, Nonito Pagès, Olivier Gros.

**Project administration:** Nonito Pagès, Olivier Gros.
**Resources:** Nonito Pagès, Olivier Gros.

**Software:** Damien F. Meyer, Rosalie Aprelon.

**Supervision:** Nonito Pagès, Olivier Gros.

**Validation:** Damien F. Meyer, Nonito Pagès, Olivier Gros.

**Visualization:** Nonito Pagès, Olivier Gros.

**Writing – original draft:** Suzanne Conjard.

**Writing – review & editing:** Suzanne Conjard, Damien F. Meyer, Nonito Pagès, Olivier Gros.

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