The First Finding of Six Instars of Larvae in Heteroptera and the Negative Correlation between Precipitation and Number of Individuals Collected in Sea Skaters of Halobates (Heteroptera: Gerridae)

This study, conducted during a scientific cruise, MR15-04, aims, first, to examine species and larval/adult components of Halobates (Heteroptera: Gerridae) inhabiting the tropical Indian Ocean of 4°00′ S–7°00′ S, 101°00′ E–103°00′ E and, second, to examine the correlative relationship between precipitation just before collection and the number of sea skaters collected in November and December 2015. Near Sumatra (50 km south-west), larvae and adults of four species of Halobates (Halobates germanes White, 1883; Halobates micans Eschscholtz, 1822; Halobates princeps White, 1883; undescribed species: Halobates sp.) were collected. Adults of an undescribed species had about a 5 mm long body in a gourd-like shape. One male adult specimen of H. princeps was collected. Body length, body width, and head width was measured in all specimens of Halobates. Six larval stages were detected in all three species of sea skaters as the first finding for Heteropteran insects. There was a negative correlation between amount of precipitation for 19 h before collection and the number of Halobates individuals collected by the neuston net. Death or (positive or passive) sinking by sea skaters could be due to occasional rain fall on the sea surface.


Introduction
Many great voyages have been launched to explore the oceans and what lies beyond, as they have always held a great fascination for us. A great variety of marine organisms were collected and described during these voyages, but insects appear to have received little attention [1]. Although they are the most abundant animals on land, insects are relatively rare in marine environments [2]. However, a few thousand insect species belonging to more than 20 orders are considered to be marine [3,4]. The majority of marine insects belong to the Coleoptera, Hemiptera, and Diptera orders, and they can be found in various marine habitats. However, the only insects to live in the open ocean are members of the genus Halobates, commonly known as sea-skaters [2]. They belong to the family Gerridae (Heteroptera), which comprises the common pond-skaters or water-striders. Unlike most of its freshwater relatives, the genus Halobates is almost exclusively marine. Adults are small, measuring only about 0.5 cm in body length, but they have rather long legs and may have a leg span of 1.0-1.5 cm [1]. Although the key to identifying the species in the genus Halobates hase shown to be morphological details of the genitalia in adult males, as noted in the appendix of Andersen and Chen [1], the larvae have not yet been described. The primary purpose of this study is to measure the body length, body width and head width of all Halobates larvae collected in this cruise and clarify the number of stages of sea skater larvae collected.
Sampling of Halobates has been performed in different geographical positions in cruises in the Pacific Ocean. However, no information has been presented on the species dynamics and individual composition in the field near the shores of the Indonesian islands. The second purpose of this study is to clarify the species component and population density of sea skaters inhabiting the area near Sumatra.
The relationship between weather conditions such as surface water temperature and population density of sea skaters has been reported. For example, the most appropriate temperature for Halobates micans and H. germanus were around 27 to 30 • C, but ranged more widely for the smaller species of H. sericeus, from 22 to 30 • C [5,6]. This preference of H. sericeus for a wider temperature range may be related to the wider range of latitudes this species inhabits. However, the possibility that rain fall is also related to the temperature range inhabited by sea skaters has not yet been examined. The third purpose of this study is to clarify this relationship in the sea skaters inhabiting the area 50 km from Sumatra.

Samplings
Samplings were performed every three days from 20 November to 14 December 2015 in the area of 4 • S-7 • S, 101 • E-103 • E with a neuston net (6 m long and diameter of 1.3 m). The neuston net was trailed for 15 mm × 3 times (3 trials) per one night on the starboard side of R/V MIRAI (8687t) owned by the Japan Agency for Marine-earth Science and TECHnology (JAMSTEC, Yokosuka, Japan). Each trailing comprised three 15-min trials at night for nine nights with the ship speed of 2.0 knot to the sea water. This was repeated twice at each station. The surface area swept by the neuston net was evaluated as an expression of (flow-meter value × 1.3 m of width of the neuston net). Precipitation was measured with radio detecting and ranging during the ship of MR15-04 on the deck of the R/V MIRAI.

Treatments of Specimens after the Samplings
Sea skaters trapped in the grey plastic bottle fixed to the end of the neuston net were paralyzed from the physical shock of the trailing of the net. Paralyzed sea skaters were transferred to a paper towel to respire. Paralysis subsided within 20 min. for most specimens. When sea skaters were trapped in the jelly of a jelly fish, the jelly was removed from the body very carefully and quickly by hand for recovery from paralysis.

Measurement of Body Sizes
Body length, body width, and head width were measured in all sea skater specimens collected during the cruise with a stereoscopic microscope, stage micrometer, and ocular micrometer. Photos of adults and larvae (H. germanus, H. micans, H. princeps and H. sp.) were taken by digital video camera (HC-V100M Panasonic; Panasonic Co Ltd., Osaka, Japan) during the cruise.

Statistic Analysis
The data was analyzed with SPSS 12.0 (12.0 J for Windows; SPSS Inc., Chicago, IL, USA) statistical software. ANCOVA and ANOVA analyses were performed on the relationship between body length, body width, and head width, species, and stage for the three species of Halobates. Body size was compared between Halobates micans and Halobates sp. with Mann-Whitney U tests for continuous values. Pearson's correlation analysis was used for the correlative analysis between precipitation and density of sea skater specimens collected.

Distribution
In samplings of Halobates (Table 1 (A-2)) inhabiting tropical stations in the eastern Indian Ocean, 12 to 330 individuals were collected per trial of four species of Halobates germanus, H. micans, H. princeps and one undescribed and relatively large species of H. sp. This undescribed species has an adult body length of about 5 cm with a gourd-like shape (Figures 1 and 2). Morphological study and precise comparison with all the 71 species described in the appendix of the key of the identification of Halobates Eschsholtz [1] indicates that this is likely a new species. Larvae and adult specimens of these four species were collected at the stations within 04 • 00 S-06 • 00 S, 101 • 00 E-103 • 00 E. The population density at Station 1 (  Table 2). At the Stations 6 and 7, 50 and 152 larvae were collected, respectively, and 51 exuviae (wasted skin at molting) were caught in total. Reproductive and growth activity might be active at these two stations.     Table 3 shows the body length, body width and head width of all larvae and adults of four species, Halobates micans, H. germanus, H. princeps and H. sp. Six instars in the larval stage were detected as the first finding of all three species (Table 3 and Figure 1). Similar to previous studies [1], all stages of larvae and adults of H. micans had a larger body length, body width, and head width than H. germanus (Tables 3 and 4). The undescribed species had a specific gourd-like shape and the body lengths of the 2nd to 5th instars larvae and adults were significantly greater than those of the other species (for example H. micans) ( Table 5).   Table 3 shows the body length, body width and head width of all larvae and adults of four species, Halobates micans, H. germanus, H. princeps and H. sp. Six instars in the larval stage were detected as the first finding of all three species (Table 3 and Figure 1). Similar to previous studies [1], all stages of larvae and adults of H. micans had a larger body length, body width, and head width than H. germanus (Tables 3 and 4). The undescribed species had a specific gourd-like shape and the body lengths of the 2nd to 5th instars larvae and adults were significantly greater than those of the other species (for example H. micans) ( Table 5).  Table 3 shows the body length, body width and head width of all larvae and adults of four species, Halobates micans, H. germanus, H. princeps and H. sp. Six instars in the larval stage were detected as the first finding of all three species (Table 3 and Figure 1). Similar to previous studies [1], all stages of larvae and adults of H. micans had a larger body length, body width, and head width than H. germanus (Tables 3 and 4). The undescribed species had a specific gourd-like shape and the body lengths of the 2nd to 5th instars larvae and adults were significantly greater than those of the other species (for example H. micans) ( Table 5).

Distribution
Reviewing the results of seven sea skater samplings performed in the tropical to subtropical Pacific Ocean and tropical Indian Ocean, Halobates micans were exclusively dominant in the tropical Indian Ocean (Table 1 (B, C), and KH-10-05 cruise: Harada et al. [13]).
In the tropical Pacific Ocean in the lower latitude area of 10 • S-10 • N, not H. micans but H. germanus occupied the area near the islands even in the lower latitude area (Table 1 (D-2, G)) [1]. In the higher latitude area of 24 • -25 • N, H. sericeus was the exclusively dominant species in the subtropical Pacific Ocean (Table 1 ( D-3, E-1, F-2)).
The difference in the species component of oceanic sea skaters due to the latitude and distance from the islands may be related to cannibalism by a bigger species (H. micans) of a smaller one (H. sericeus) (Harada, unpublished), tolerance to variation in surface temperature [1,14,15] (H. sericeus inhabiting a wider range of latitudes and with harder tolerance to temperature change) and salinity [16] (H. germanus with a harder tolerance to lower salinity because of selection by heavy rain fall from inhabiting the area near the shore).

Larval Development of Larvae of Sea Skaters
Most of the species included in Heteroptera, Insecta have five stages of larvae, for example the chinch bug, Blissusleu copterus, and the harlequin bug, Murgantia histrionic [17]. The measurements of body length, body width, and head width were performed in all larvae and adults collected in this cruise (MR15-04) by the R/V MIRAI in November-December 2016. This measurement made it clear that the number of larvae was not five but six because of the existence of 0th instars before the normal five instars. The cruise of MR06-04 by the R/V MIRAI was performed in the western tropical Pacific Ocean. More than 3000 eggs that had been laid on a piece of styrene foam were collected by an ORI (Ocean Research Institute of the University of Tokyo produced) net during this cruise [14]. These eggs were incubated and most 0th instar larvae hatched from the eggs [18]. However, none molted out into the next 1st instar larvae, despite careful incubation with an appropriate temperature of around 30 • C and sufficient food (larvae fish collected by the ORI and neuston nets and adult Calliphoridae flies, Lucillia irrustris). At least larvae of the 3rd to 5th instars had been observed to molt into the next stage during incubation in the 9 cruises by the R/V MIRAI [18]. The molt from 0th instar to 1st instar was not successful during incubation on the ships previously [19]. Specific conditions including food, temperature, PH, and lighting may be critical for development and/or molting of the 0th instar. Because of this difficulty of incubation, perfect and circular incubation throughout the life cycle of sea skaters has not been possible.
The most apparent characteristic of the undescribed species inhabiting the off-shore area (50 km away from the shore) of Sumatra was the relative long body length in the last instar (5th instar and adult stage).
The characteristic of elongated body form may have developed on the way to evolutionary differentiation from other Halobates species relatively recently within the last 100 million years due to the famous Haeckel theory of phylogeny and ontogeny development [20], because the development of the body lengths of 0th to 3rd instars are similar to that of another similar-sized oceanic sea skater, H. micans.

Conclusions
Larvae and adults of four species of Halobates (H. germanus, H. micans, H. micans and undescribed species: H. sp.) were collected 50 km south-west of Sumatra. Adults of the undescribed species were characterized by a gourd-shaped body that was more than 5 mm long and longer than the other oceanic sea skaters we collected. There was a negative correlation between the amount of precipitation 19 h before collection and the number of Halobates specimens collected with a neuston net. The lower number of sea skaters collected could be associated with a drop in salinity and/or lower temperatures attributed to rain squalls.