Clunio marinus Haliday, 1855

Michailova, Paraskeva, 2022, External morphology of larva and polytene chromosomes of Clunio marinus Haliday, 1855 (Diptera, Chironomidae, Orthocladiinae) from two localities of the Atlantic coast, Zootaxa 5141 (2), pp. 163-173 : 164-172

1. Larva of Clunio marinus Haliday , lab. stock, Vigo, Spain

Small sized species,length up to 5 mm long. General coloration yellowish brown, head dark brown. Mentum with dark brown teeth, mandible with brown teeth. Mandible is about 130 µm long and about 44 µm width ( Fig. 1A ) with 5 teeth. Seta subdentalis triangular, reaching almost half way up the last inner tooth which is lighter than the others; seta interna has 5 strong branches. Mentum ( Fig. 1B ): median tooth rounded and smooth, its width (W) is recorded in Table 1 ; the four lateral teeth are rounded apically and decreasing in size from 1 to 4. Ventromental plates poorly developed, seta submenti about 22 µm long, quite prominent and not branched; distance (D1) between both setae submenti can see in Table 1 . Antenna ( Fig. 1C ): 5 segments, lengths of segments L1 and L2 as in Table 1 . Segment 1 with Ring Organ (RO) located at upper part of the segment 1. Segment 1 with a blade (bl) consisting of two parts, reaching apex of 4 th segment. Segment 2 with style (st) reaching the apex of 3 rd segment. Segments 3 and 4 almost sub-equal. Lauterborn organ very weak, difficult to see. Premandible (Pm) ( Fig. 1D , 2A, 2C ): bare, with a large blunt slightly prolonged tooth and an inner rounded blunt tooth. The proximal part of premandible is not broarder than the distal part. Pecten epipharyngis (PE) ( Fig. 2A ): consists of non-fused structures at base and lanceolate setae on both sides. Maxilla ( Fig. 2B ): has a well developed maxillary palp (mp), with a single seta, located laterally, lacinia with several simple maxillary chaetae (LCh). Posterior parapods (pp) ( Fig. 2D ): about 60 µm long, anal setae (as) difficult to see, supra-anal setae (ss) 120-122 µm long, ventral tubules absent. Clypeus light, almost trapezoidal with weak granulation on both sides near to the posterior margin; setae I and II plumose ( Fig. 2C ), occasionally with branches; setae III and IV simple. Setae S1 located close to the anterior margin ( Figs. 2C , 3A ); setae S2 inserted laterally ( Fig. 3A ). Frontal apotome dark brown, elongated ( Fig. 3A ); setae S3-S5 located near the lateral margin; S3, close to the anterior margin; S4, antero-medially; S5, on median area.

2. Larva of Clunio marinus Haliday , lab. stock, Helgoland, Germany

Small sized species, length up to 6 mm long. General coloration yellowish green, head dark brown. Mentum with dark brown teeth, mandible with brown teeth. Mandible ( Fig. 4A ): with 5 teeth, about 124 µm long and about 37 µm wide. Seta subdentalis triangular, reaching the tip of the last inner tooth; seta interna has 5 distinct branches. There are also C. marinus larvae in the analyzed material in which the seta subdentalis reaches the middle part of the last tooth. Mentum ( Fig. 4B ): dark, the tip of middle tooth, is more pointed than the rounded tooth observed at Vigo, its width (W) is recorded in Table 2 ; the four lateral teeth are pointed apically and decrease in size from 1 to 4. Ventromental plates (v.p) poorly developed, seta submenta about 26 µm long, distinct and not branched; distance between the setae submenti is in Table 2 . Antenna ( Fig. 4C ): 5 segments, lengths of segments L1 and L2 as in Table 2 . Segment 1 with Ring Organ (RO) located at upper part of the segment; segment 1 with a blade (bl) consisting of two parts, reaching proximal half of 4 th segment; segment 2 with style (st) reaching one third of the fourth segment; 4 th segment longer than the 3th one; lauterborn organ very weak, difficult to see. Premandible (Pm) ( Fig. 4D , 5A ): bare, with a blunt tooth and broad, blunt, rounded inner tooth. The proximal part of premandible is not broarder than the distal region. Pecten epipharyngis (PE) ( Fig. 5A ): consists of non-fused structures at base and lanceolate setae on both sides. Maxilla ( Fig. 5B ): has a well developed maxillary palp (mp), with a single seta, located laterally, lacinia with several simple maxillary chaetae (LCh). Posterior parapods (pp) ( Fig. 5C ): about 80 µm. Near to the posterior parapods there are anal setae (as) 60 µm long, and strong supra-anal setae (ss) 160 µm long, ventral tubules absent. Clypeus trapezoidal with wide posterior margin, dense granulation on both sides ( Fig. 6A ); setae I and II plumose ( Fig. 5A ), occasionally with branches; setae III and IV simple. Setae S1 located close to the anterior margin, setae S2 inserted laterally ( Figs. 5A , 6A ). Frontal apotome dark brown, elongated ( Fig. 6A ): setae S3-S5 located near the lateral margin; S3, close to the anterior margin; S4, antero-medially; S5, on median area.

3. Polytene chromosomes of Clunio marinus Haliday , lab. stock, Vigo, Spain

The chromosome set is 6. The characteristic sign of the species polytene chromosomes is a very low degree of polyteny and poor band structure. Very often pairing between different parts of one and the same chromosome is established. The position of centromeres is not possible to determine, the difficult spreading of the chromosome is obvious. The first chromosome carries a Nucleolar Organizer (NOR) and a Balbiani ring (BR) ( Fig. 7A ). The chromosome near to the Nucleolar Organizer (NOR) has a funnel-shaped extension, followed by a dark band, a marker of the chromosome. A dark band, indicated by arrow, defines a third chromosome ( Fig. 7A ). A complex heterozygous inversion, found in 50% of the studied individuals, involving the telomere of the second chromosome was found. A complex heterozygous inversion (75%) was recorded on the third chromosome in three individuals ( Figs. 7A, B ). Both inversions were also found in the species from Helgoland ( Michailova 1980). However, due to the lack of a clear band structure of the species from Vigo, it was not possible to make a detailed comparative cytogenetic analysis with C. marinus from Helgoland. It is possible to have cytogenetic differentiation, shown by fixed homozygous inversions, as has been found in species of the genus Chironomus Meigen (1803) , or different populations of the same species of that genus ( Kiknadze et al. 2016).

When we compare the external morphology of the larvae with those of other species we found the following differences: Median tooth of mentum is rounded and smooth ( Fig. 1B , this study), triangular apically in C. balticus (Moubayed-Breil & Michailova 2020, Fig. 3B ), sub-triangular to rounded apically in C. marinus ( Strenzke 1960, Fig. 31), semi-circular in C. mediterraneus ( Tasdemir 2010, Fig. 4 ), sub-trapezoidal in C. sp. 1 ( Abdelsalam 2017, Fig. 5 ); widely domed and rounded in C. ponticus (Moubayed-Breil et al. 2020, Fig. 11B); Seta subdentalis triangular, reaching almost the middle part of the last tooth; or triangular, reaching the tip of the last tooth ( Figs. 1A and 4A , this study). The seta subdentalis long and sharpened to the apex, reaching the last (posterior) tooth as in C.balticus (Moubayed-Breil & Michailova 2020, Fig. 3A ); C. ponticus (Moubayed-Breil et al. 2020, Fig. 11A) and C. marinus ( Strenzke 1960, Fig. 29), C. sp. 1 ( Abdelsalam 2017, Fig. 8). Blade, reaching apex of 4 th segment and it’s style reaching the apex of 3 rd segment ( Fig. 1C , this study), or reaching proximal half of 4 th segment; segment 2 with style reaching one third of the fourth segment ( Fig. 4C , this study); it reaches one third of the fourth segment and the style reaches the base of segment 4 in C. balticus (Moubayed- Breil & Michailova 2020, Fig. 3C ); reaching the apex of 4 th antennal segment and the style reaching end of segment 4 in C. ponticus (Moubayed-Breil et al. 2020, Fig. 11C), reaching proximal half of 4 th segment in C. marinus and C. sp.1 ( Strenzke 1960, Fig. 2 ; Abdelsalam 2017, Fig. 5 ), reaching segment 5 in C. mediterraneus ( Tasdemir 2010, Fig. 3 ); Clypeus light, almost trapezoidal with weak granulation on both sides near to the posterior margin ( Fig. 3A , this study) or with distinct granulation on both sides to the posterior margin ( Fig. 6A , this study), trapezoidal with weak granulation present close to the area of S 2 in C. balticus (Moubayed-Breil & Michailova 2020, Fig. 4A ), trapezoidal with wide posterior margin, dense granulation present on both sides in C. ponticus (Moubayed-Breil et al. 2020, Fig. 10); anal setae of parapods difficult to see ( Fig. 2D , this sudy), distinct in C. balticus (Moubayed-Breil & Michailova 2020, Fig. 4D ), in C. ponticus (Moubayed-Breil et al. 2020, Fig. 12C), in C. marinus ( Strenzke 1960, Fig. 32), C. mediterraneus ( Tasdemir 2010, Fig. 4 ) and C. sp. 1 ( Abdelsalam 2017, Fig. 4 ).

The presented comparative analysis on the external morphology of the larva of different species of genus Clunio shows species-specific differences. This analysis shows a difference in the morphology of the mentum, in the length of seta-subdentalis of the mandible, in the length of antennal blade and style, the length of the antennal segments, the granulation on the clypeus, the reduced anal setae of parapods.

Our data from the cytogenetic analysis showed that individuals of C. marinus of the Spanish population have the same diploid chromosome set as those from Helgoland. Also, they have a well expressed Nucleolar Organizer (NOR) and Balbiani ring (BR) located in the same chromosome. However, it is difficult to carry out a comparative analysis of band patterns of the polytene chromosome of individuals from both localities due to poor band patterns of individuals from Spain. The material for cytogenetic analysis is 20 generations old, which affects the band structure of the polytene chromosomes. During the prolonged culturing, cell physiology and morphology of polytene chromosomes could be changed significantly ( Zhimulev 1996). Kaiser et al. (2010), using mitochondrial and nuclear DNA approaches, established that the nuclear genome is less differentiated. The mitochondrial genome is much more differentiated between different regions, but not within the regions. They found no Cytochrome C oxidase I (COI) haplotypes shared among regions and suggested that the major barrier of gene flow between the regions is the geographic isolation. Important for this isolation is also the differences in emergence time of the adults between regions and local timing adaptations. These authors’ emphase that the long sandy stretches between the regions are additional strong geographic barriers to gene flow. According to the authors, larval dispersal between Europe’s major rocky coasts is probably an extremely rare event.

Our analysis of larval morphology from two geographically distant regions ( Spain and Germany) shows external morphological differentiation, which affected the morphology of the larva, including the morphology of the mentum, the length of antennal blade and stylus, the length of 3 rd and 4 th antennal segments and the distance between mentum setae and the granulation on the clypeus. The larvae are adapted to the local conditions and they have constant population larval characters. The reason for this differentiation might lie in the peculiarities of C. marinus biology. Females are wingless and males live a very short time. This limits gene flow between distant populations and hence each population evolves independently of the others. Every population adapts to the specific environmental conditions and the resulting changes affect different systems and structures. In this study, changes are observed at the morphological level of the larval stage. Kaiser’s et al. (2010) studies on C. marinus from these populations showed differentiation in the mitochondrial genome and there may be differentiation at the cytogenetic level also, mainly the presence of homozygous inversions as post-mating isolating mechanisms. However, the poor structure of the polytene chromosomes of the Spanish population does not allow a detailed comparative cytogenetic analysis with the Helgoland population. Our study shows differentiation at the morphological level, which affects the external morphology of the larva. Therefore, there is a differentiation at different levels of biological organization, both at the morphological and at the molecular genetic level.

Figures

FIGURE 1. Larva of Clunio marinus Holiday, lab. stock, Spain, Vigo. A. Mandible; B. Mentum with median and lateral teeth; C. Antenna: blade (bl), style (st); D. Premandible (Pm). Scale bar, 10 µm.

FIGURE 2. Larva of Clunio marinus Holiday, lab.stock, Spain, Vigo.A. Pecten epipharyngis (PE), premandible (Pm); B. Maxilla with maxillary palp (mp) and lacinial chaetae (LCh); C. Clypeus with setae SI and SII and premandible (Pm); D. Caudal part with posterior parapods (pp), anal seta (as), and supra anal setae (ss). Scale bar, 10 µm.

FIGURE 3. Larvae of Clunio marinus Holiday, lab. stock, Spain, Vigo. A. Clypeus and frontal apotome with setae S1 to S5. Scale bar, 10 µm.

FIGURE 4. Larva of Clunio marinus Holiday, lab. stock, Germany, Helgoland. A. Mandible; B. Mentum with median and lateral teeth; C. Antenna: blade (bl), style (st); D. Premandible (Pm). Scale bar, 10 µm.

FIGURE 5. Larva of Clunio marinus Holiday, lab. stock, Germany, Helgoland. A. Pecten epipharyngis (PE) and premandible (Pm); B. Maxilla with maxillary palp (mp) and lacinial chaetae (LCh); C. Caudal part with posterior parapods (pp), anal seta (as) and supra anal seta (ss). Scale bar, 10 µm.

FIGURE 6. Larvae of Clunio marinus Holiday, lab. stock, Germany, Helgoland. A. Clypeus and frontal apotome with setae S3 to S5. Scale bar, 10 µm.

FIGURE 7. A. Polytene chromosomes: I, II, III of Clunio marinus Holiday, lab. stock, Spain, Vigo. Heterozygous inversion in chromosome III. Arrows indicate the markers of the chromosomes I and III. NOR—Nucleolar Organizer; BR—Balbiani Ring. B. Heterozygous inversion in chromosome III. Scale bar, 10 µm.